Regulation of lung tissue by hedgehog-like polypeptides, and formulations and uses related thereto

ABSTRACT

The present application relates to a method for modulating the growth state of an lung tissue, or a cell thereof, e.g., by ectopically contacting the tissue, in vitro or in vivo, with a hedgehog therapeutic, a ptc therapeutic, or an FGF-10 therapeutic in an amount effective to alter the rate (promote or inhibit) of proliferation of cells in the lung tissue, e.g., relative to the absence of administration of the hedgehog therapeutic or ptc therapeutic. The subject method can be used, for example, to modulate the growth state of epithelial and/or mesenchymal cells of a lung tissue, such as may be useful as part of a regimen for prevention of a disease state, or in the treatment of an existing disease state or other damage to the lung tissue.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional application60/099,952, filed Sep. 11, 1998 and entitled “Regulation of Lung Tissueby Hedgehog-like Polypeptides, and Formulations and Uses RelatedThereto”, the specification of which is incorporated by referenceherein.

GOVERNMENT FUNDING

Certain work described herein was funded by the National Institutes ofHealth. The government may have rights in inventions described herein.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homoiogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis.

The first hedgehog gene was identified by a genetic screen in thefruitfly Drosophila melanogaster (Nüsslein-Volhard, C. and Wieschaus, E.(1980) Nature 287, 795-801). This screen identified a number ofmutations affecting embryonic and larval development. In 1992 and 1993,the molecular nature of the Drosophila hedgehog (hh) gene was reported(C. F., Lee et al. (1992) Cell 71, 33-50), and since then, severalhedgehog homologues have been isolated from various vertebrate species.While only one hedgehog gene has been found in Drosophila and otherinvertebrates, multiple Hedgehog genes are present in vertebrates.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal. (1994) Science 266:1528-1537; Porter et al. (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al.(1992) supra; Chang et al. (1994) supra; Lee et al. (1994) supra;Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter etal. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai,C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptidestays tightly associated with the surface of cells in which it wassynthesized, while the C-terminal peptide is freely diffusible both invitro and in vivo (Lee et al. (1994) supra; Bumcrot et al. (1995) supra;Mart', E. et al. (1995) Development 121:2537-2547; Roelink, H. et al.(1995) Cell 81:445-455). Interestingly, cell surface retention of theN-terminal peptide is dependent on autocleavage, as a truncated form ofHH encoded by an RNA which terminates precisely at the normal positionof internal cleavage is diffusible in vitro (Porter et al. (1995) supra)and in vivo (Porter, J. A. et al. (1996) Cell 86, 21-34). Biochemicalstudies have shown that the autoproteolytic cleavage of the HH precursorprotein proceeds through an internal thioester intermediate whichsubsequently is cleaved in a nucleophilic substitution. It is likelythat the nucleophile is a small lipophilic molecule which becomescovalently bound to the C-terminal end of the N-peptide (Porter et al.(1996) supra), tethering it to the cell surface. The biologicalimplications are profound. As a result of the tethering, a high localconcentration of N-terminal Hedgehog peptide is generated on the surfaceof the Hedgehog producing cells. It is this N-terminal peptide which isboth necessary and sufficient for short and long range Hedgehogsignaling activities in Drosophila and vertebrates (Porter et al. (1995)supra; Ekker et al. (1995) supra; Lai et al. (1995) supra; Roelink, H.et al. (1995) Cell 81:445-455; Porter et al. (1996) supra; Fietz, M. J.et al. (1995) Curr. Biol. 5:643-651; Fan, C.-M. et al. (1995) Cell81:457-465; Mart', E., et al. (1995) Nature 375:322-325; Lopez-Martinezet al. (1995) Curr. Biol 5:791-795; Ekker, S. C. et al. (1995)Development 121:2337-2347; Forbes, A. J. et al. (1996) Development122:1125-1135).

HH has been implicated in short- and long e range patterning processesat various sites during Drosophila development. In the establishment ofsegment polarity in early embryos, it has short range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long range effects via the induction of secondarysignals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears (see Table 1). Of these genes, Shh has received most of theexperimental attention, as it is expressed in different organizingcenters which are the sources of signals that pattern neighbouringtissues. Recent evidence indicates that Shh is involved in theseinteractions.

The interaction of a hedgehog protein with one of its cognate receptor,patched, sets in motion a cascade involving the activation andinhibition of downstream effectors, the ultimate consequence of whichis, in some instances, a detectable change in the transcription ortranslation of a gene. Transcriptional targets of hedgehog signaling arethe patched gene itself (Hidalgo and Ingham, 1990 Development 110,291-301; Marigo et al., 1996) and the vertebrate homologs of thedrosophila cubitus interruptus (Ci) gene, the GLI genes (Hui et al.(1994) Dev Biol 162:402-413). Patched gene expression has been shown tobe induced in cells of the limb bud and the neural plate that areresponsive to Shh. (Mango et al. (1996) Development 122:1225-1233). TheGLI genes encode putative transcription factors having zinc finger DNAbinding domains (Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzleret al. (1990) Mol Cell Biol 10:634-642). Transcription of the GLI genehas been reported to be upregulated in response to hedgehog in limbbuds, while transcription of the GLI3 gene is downregulated in responseto hedgehog induction (Marigo et al. (1996) Development 122:1225-1233).Moreover, it has been demonstrated that elevated levels of Claresufficient to activate patched (ptc) and other hedgehog target genes,even in the absence of hedgehog activity.

SUMMARY OF THE INVENTION

One aspect of the present application relates to a method for modulatingthe growth state of an lung tissue, or a cell thereof, e.g., byectopically contacting the tissue, in vitro or in vivo, with a hedgehogtherapeutic, a ptc therapeutic, or an FGF-10 therapeutic (describedinfra) in an amount effective to alter the rate (promote or inhibit) ofproliferation of cells in the lung tissue, e.g., relative to the absenceof administration of the hedgehog therapeutic or ptc therapeutic. Thesubject method can be used, for example, to modulate the growth state ofepithelial and/or mesenchymal cells of a lung tissue, such as may beuseful as part of a regimen for prevention of a disease state, or in thetreatment of an existing disease state or other damage to the lungtissue.

Wherein the subject method is carried out using a hedgehog therapeutic,the hedgehog therapeutic preferably a polypeptide including a hedgehogportion comprising at least a bioactive extracellular portion of ahedgehog protein, e.g., the hedgehog portion includes at least 50, 100or 150 (contiguous) amino acid residues of an N-terminal half of ahedgehog protein. In preferred embodiments, the hedgehog portionincludes at least a portion of the hedgehog protein corresponding to a19 kd fragment of the extracellular domain of a hedgehog protein.

In certain preferred embodiments, the hedgehog portion has an amino acidsequence at least 60, 75, 85, or 95 percent identical with a hedgehogprotein of any of SEQ ID Nos. 10-18 or 20, though sequences identical tothose sequence listing entries are also contemplated as useful in thepresent method. The hedgehog portion can be encoded by a nucleic acidwhich hybridizes under stringent conditions to a nucleic acid sequenceof any of SEQ ID Nos. 1-9 or 19, e.g., the hedgehog portion can beencoded by a vertebrate hedgehog gene, especially a human hedgehog gene.

In certain embodiments, the hedgehog polypeptide is modified with one ormore sterol moieties, e.g., cholesterol or a derivative thereof.

In certain embodiments, the hedgehog polypeptide is modified with one ormore fatty acid moieties, such as a fatty acid moiety selected from thegroup consisting of myristoyl, palmitoyl, stearoyl, and arachidoyl.

In other embodiments, the subject method can be carried out byadministering a gene activation construct, wherein the gene activationconstruct is deigned to recombine with a genomic hedgehog gene of thepatient to provide a heterologous transcriptional regulatory sequenceoperatively linked to a coding sequence of the hedgehog gene.

In still other embodiments, the subject method can be practiced with theadministration of a gene therapy construct encoding a hedgehogpolypeptide. For instance, the gene therapy construct can be provided ina composition selected from a group consisting of a recombinant viralparticle, a liposome, and a poly-cationic nucleic acid binding agent,

In yet other embodiments, the subject method can be carried out using aptc therapeutic. An exemplary ptc therapeutic is a small organicmolecule which binds to a patched protein and derepressespatched-mediated inhibition of mitosis, e.g., a molecule which binds topatched and mimics hedgehog-mediated patched signal transduction, whichbinds to patched and regulates patched-dependent gene expression. Forinstance, the binding of the ptc therapeutic to patched may result inupregulation of patched and/or gli expression.

In a more generic sense, the ptc therapeutic can be a small organicmolecule which induces hedgehog-mediated patched signal transduction,such as by altering the localization, protein-protein binding and/orenzymatic activity of an intracellular protein involved in a patchedsignal pathway. For instance, the ptc therapeutic may alter the level ofexpression of a hedgehog protein, a patched protein or a proteininvolved in the intracellular signal transduction pathway of patched.

In certain embodiments, the ptc therapeutic is an antisense constructwhich inhibits the expression of a protein which is involved in thesignal transduction pathway of patched and the expression of whichantagonizes hedgehog-mediated signals. The antisense construct ispreferably an oligonucleotide of about 20-30 nucleotides in length andhaving a GC content of at least 50 percent.

In other embodiments, the ptc therapeutic is an inhibitor of proteinkinase A (PKA), such as a 5-isoquinolinesulfonamide. The PKA inhibitorcan be a cyclic AMP analog. Exemplary PKA inhibitors includeN-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide,1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, KT5720, 8-bromo-cAMP,dibutyryl-cAMP and PKA Heat Stable Inhibitor isoform α. Anotherexemplary PKA inhibitor is represented in the general formula:

wherein,

R₁ and R₂ each can independently represent hydrogen, and as valence andstability permit a lower alkyl, a lower alkenyl, a lower alkynyl, acarbonyl (such as a carboxyl, an ester, a formate, or a ketone), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₈, or

R₁ and R₂ taken together with N form a heterocycle (substituted orunsubstituted);

R₃ is absent or represents one or more substitutions to the isoquinolinering such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₈;

R₈ represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and

n and m are independently for each occurrence zero or an integer in therange of 1 to 6.

The subject method can be used to prevent or treat various lungdiseases, to control wound healing or other reformation processes inlung, and to augment lung transplantation.

Wherein the subject method is carried out using an fgf-10 therapeutic,the fgf-10 therapeutic preferably a polypeptide including a fgf-10portion comprising at least a bioactive extracellular portion of afgf-10 protein, e.g., the fgf-10 portion includes at least 50, 100 or150 (contiguous) amino acid residues of a fgf-10 protein, preferably ahuman fgf-10 protein such as shown in SEQ ID No. 24.

In certain preferred embodiments, the fgf-10 portion has an amino acidsequence at least 60, 75, 85, or 95 percent identical with the fgf-10protein of SEQ ID No. 24, though a sequence identical with SEQ ID No. 24is also contemplated as useful in the present method. The fgf-10 portioncan be encoded by a nucleic acid which hybridizes under stringentconditions to a nucleic acid sequence of SEQ ID No. 23, e.g., the fgf-10portion can be encoded by a vertebrate fgf-10 gene, especially a humanfgf-10 gene.

In other embodiments, the subject method can be carried out byadministering a gene activation construct, wherein the gene activationconstruct is deigned to recombine with a genomic fgf-10 gene of thepatient to provide a heterologous transcriptional regulatory sequenceoperatively linked to a coding sequence of the fgf-10 gene.

In still other embodiments, the subject method can be practiced with theadministration of a gene therapy construct encoding a fgf-10polypeptide. For instance, the gene therapy construct can be provided ina composition selected from a group consisting of a recombinant viralparticle, a liposome, and a poly-cationic nucleic acid binding agent,

Yet another aspect of the present invention concerns preparations of ahedgehog, ptc or fgf-10 therapeutic formulated for application to lungtissue, e.g., by aerosol. For example, such formulations may include apolypeptide comprising a hedgehog polypeptide sequence including abioactive fragment of a hedgehog protein, which polypeptide isformulated for application to lung tissue by inhalation.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1. Morphology and epithelial phenotype of Shh −/− mouse lungs. (a)At 12.5 dpc, the wt mouse lung has branched several times to give riseto distinct lobes (arrows). (b) Trachea and esophagus are separatetubes. (c) Cross-section at the level of the lung shows branching andlobation. (d) At 12.5 dpc, Shh-deficient lungs have failed to undergolobation or subsequent extensive branching. (e) Trachea and esophagusremain fused at the tracheoesophageal septum. (f) Mutant lungs havebranched only once. (g) At 18.5 dpc, airsac formation is in progress inthe wt and the respiratory surface is in tight association with bloodvessels. (h) There is little branching or growth of the poorlyvascularized mutant lungs, but airsac formation at the distal epithelialtips is apparent (arrows). (i) By 18.5 dpc, wild-type lungs haveestablished the conducting airways and respiratory bronchioles, alveolarformation is in progress. (j) In contrast, in a mutant lung of the samestage, branching is dramatically decreased. Only a few primary branches(arrows) and air sacs (arrowheads) are present. (k) In the wild-type,trachea and esophagus are separated. The trachea is lined by columnarcells, the esophagus by stratified epithelium. (l) Air sacs are made ofcuboidal cells. (m) In the mutant, trachea and esophagus are fused toform a fistula. Differentiation into columnar and stratified epitheliumis apparent, (n) as is the characteristic cuboidal epithelium of the airsacs. Demarcation lines between terminal bronchioles and respiratorysurface are indicated. (o) Proximal lung epithelium of the 18.5 dpc wtlung expresses CCSP in Clara cells, and (p) SP—C in type II pneumocytesof the distal epithelium. (q) CCSP and (p) SP—C are expressed in thecorrect proximo-distal domain in the mutant. Bars denote 1 mm (g,h only)or 10 μm. (a,d,g,h) are ventral views, all others transverse sections.Abbreviations: t—trachea, e—esophagus, l—lung, h—heart, s—stomach,mb—mainstem bronchus, b—bronchus, tb—terminal bronchiole, a—air sac.

FIG. 2. In situ analysis of gene expression in the lungs of Shh mutants.Expression of the genes indicated was investigated in whole mountvibratome sections through lungs removed from wt 11.5 and 12.5 dpc, andShh-mutant 12.5 dpc embryos.

FIG. 3. Mesenchyme differentiation at 18.5 dpc. (a) Both wt and mutantlungs display cartilaginous rings around the trachea as indicated byalcian-blue staining. (b) While to in the wild-type lung a layer ofsmooth muscle surrounds the conducting epithelium, the mutant lungmesenchyme does not differentiate into muscle (right panel). Bars denote10 mm

DETAILED DESCRIPTION OF THE INVENTION

Development of the lung, through a process known as branchingmorphogenesis, is strictly dependent on interactions betweenendodermally derived epithelial cells and the splanchnic mesenchyme.Cell-cell interactions form the functional basis for branchingmorphogenesis and occur through the activity of a number of mediators,including the extracellular matrix, cellular receptors, andmorphogenetic signaling molecules such as peptide growth factors. Themolecular regulatory signals and in particular the role oftranscriptional factors in branching morphogenesis and lunginjury/repair are an important source of information for the treatmentof injury. Furthermore, because the lungs continue to undergodevelopment after birth, untimely activation of alternativemorphogenetic signals released by tissue injury or repair or both maypotentially derail normal morphogenesis and result in structural andfunctional aberrations characteristic of neonatal lung disease.

It is demonstrated herein that hedgehog proteins, such as Shh, isessential for development of the respiratory system. In Shh nullmutants, for example, the trachea and esophagus do not separate properlyand the lungs form a rudimentary sac due to failure of branching andgrowth after formation of the primary lung buds. Interestingly, normalproximo-distal differentiation of the airway epithelium occurs,indicating that Shh is not needed for differentiation events. Inaddition, the transcription of several mesenchymally expresseddownstream targets of Shh is abolished. These results highlight theimportance of epithelially derived Shh in regulating branchingmorphogenesis of the lung, and establish a role for hedgehog in lungmorphogenesis, disease and repair, and suggest that SHH normallyregulates lung mesenchymal cell proliferation in vivo.

I. Overview

The present application is directed to the discovery that preparationsof hedgehog polypeptides can be used to control the formation and/ormaintenance of lung tissue. As described in the appended examples,hedgehog proteins are implicated in the proliferation anddifferentiation of lung mesenchymal and epithelial cells and provideearly signals that regulate the formation and maintenance of lungtissues. The present invention provides a method for regulating thegrowth state of lung tissue, e.g., either in in vitro or in vivo. Ingeneral, the method of the present invention comprises contacting lungtissue, or cells derived therefrom, with an amount of a hedgehogtherapeutic (defined infra) which produces a non-toxic response by thecell of induction or inhibition of the formation of lung tissuemicroarchetecture, e.g., depending on the whether the hedgehogtherapeutic is a sufficient hedgehog agonist or hedgehog antagonist. Thesubject method can be carried out on lung cells which may be eitherdispersed in culture or a part of an intact tissue or organ. Moreover,the method can be performed on cells which are provided in culture (invitro), or on cells in a whole animal (in vivo).

Without wishing to be bound by any particular theory, the ability ofhedgehog proteins to regulate the growth state of lung tissue may be dueat least in part to the ability of these proteins to antagonize(directly or indirectly) patched-mediated regulation of gene expressionand other physiological effects mediated by that protein. The patchedgene product, a cell surface protein, is understood to signal through apathway which causes transcriptional repression of members of the Wntand Dpp/BMP families of morphogens, proteins which impart positionalinformation. In development of the CNS and patterning of limbs invertebrates, the introduction of hedgehog relieves (derepresses) thisinhibition conferred by patched, allowing expression of particular geneprograms.

Recently, it has been reported that mutations in the human version ofpatched, a gene first identified in a fruit fly developmental pathway,cause a hereditary skin cancer and may contribute to sporadic skincancers. See, for example, Hahn et al. (1996) Cell 86:841-851; andJohnson et al. (1996) Science 272:1668-1671. The demonstration thatnevoid basal-cell carcinoma (NBCC) results from mutations in the humanpatched gene provided an example of the roles patched plays inpost-embryonic development. These observations have led the art tounderstand one activity of patched to be a tumor suppressor gene, whichmay act by inhibiting proliferative signals from hedgehog. Ourobservations set forth below reveal potential new roles for thehedgehog/patched pathway in maintenance of proliferation anddifferentiation of lung tissue. Accordingly, the present inventioncontemplates the use of other agents which are capable of mimicking theeffect of the hedgehog protein on patched signalling, e.g., as may beidentified from the drug screening assays described below.

Moreover, we demonstrate that fgf-10 is an important component of thehedgehog regulatory network present in the embryonic lung, controllingproliferation, differentiation and pattern formation. Accordingly,Applicants contemplate that agonists and antagonist of fgf-10 activity.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “hedgehog therapeutic” refers to various forms of hedgehogpolypeptides, as well as peptidomimetics, which can modulate theproliferation/differentiation state of lung cells by, as will be clearfrom the context of individual examples, mimicking or potentiating(agonizing) or inhibiting (antagonizing) the effects of anaturally-occurring hedgehog protein. A hedgehog therapeutic whichmimics or potentiates the activity of a wild-type hedgehog protein is a“hedgehog agonist”. Conversely, a hedgehog therapeutic which inhibitsthe activity of a wild-type hedgehog protein is a “hedgehog antagonist”.

In particular, the term “hedgehog polypeptide” encompasses preparationsof hedgehog proteins and peptidyl fragments thereof, both agonist andantagonist forms as the specific context will make clear.

As used herein the term “bioactive fragment of a hedgehog protein”refers to a fragment of a full-length hedgehog polypeptide, wherein thefragment specifically agonizes or antagonizes inductive events mediatedby wild-type hedgehog proteins. The hedgehog biactive fragmentpreferably is a soluble extracellular portion of a hedgehog protein,where solubility is with reference to physiologically compatiblesolutions. Exemplary bioactive fragments are described in PCTpublications WO 95/18856 and WO 96/17924.

The term “patched” or “ptc” refers to a family of related transmembraneproteins which have been implicated in the signal transduction inducedby contacting a cell with a hedgehog protein. For example, the mammalianptc family includes ptc1 and ptc2. In addition to references set outbelow, see also Takabatake et al. (1997) FEBS Lett 410:485 and GenBankAB000847 for examples of ptc2. Unless otherwise evident from thecontext, it will be understood that embodiments described in the contextof ptc1 (or just ptc) also refer to equivalent embodiments involvingother ptc homologs like ptc2.

The term “ptc therapeutic” refers to agents which either (i) mimic theeffect of hedgehog proteins on patched signalling, e.g., whichantagonize the cell-cycle inhibitory activity of patched, or (ii)activate or potentiate patched signalling. In other embodiments, the ptctherapeutic can be a hedgehog antagonist. The ptc therapeutic can be,e.g., a peptide, a nucleic acid, a carbohydrate, a small organicmolecule, or natural product extract (or fraction thereof).

The term “fgf-10 therapeutic” refers to agents which mimic orantagonize, as appropriate, the effect of fgf-10 proteins onproliferation and differentiation of lung tissue. Such agents alsoinclude small organic molecules which bind to the fgf-10 receptor andeither inhibit or agonize fgf-10 signalling.

A “proliferative” form of a ptc, hedgehog or fgf-10 therapeutic is onewhich induces proliferation of lung cells, e.g., directly or indirectly,mesenchymal or epithelial cells. Conversely, an “antiproliferative” formof a ptc, hedgehog or fgf-10 therapeutic is one which inhibitsproliferation of lung cells, preferably in a non-toxic manner, e.g., bypromoting or maintaining a differentiated phenotype or otherwisepromoting quiescence.

By way of example, though not wishing to be bound by a particulartheory, proliferative hedgehog polypeptide will generally be a form ofthe protein which derepresses patched-mediated cell-cycle arrest, e.g.,the polypeptide mimics the effect of a naturally occurring hedgehogprotein effect on lung tissues. A proliferative ptc therapeutic includesother agents which depress patched-mediated cell-cycle arrest, and mayact extracellularly or intracellularly.

An illustrative antiproliferative ptc therapeutic agent may potentiatepatched-mediated cell-cycle arrest. Such agents can be small moleculesthat inhibit, e.g., hedgehog binding to patched, as well as agents whichstimulate and/or potentiate a signal transduction pathway of the patchedprotein.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

As used herein, “transformed cells” refers to cells which havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

An “effective amount” of, e.g., a hedgehog therapeutic, with respect tothe subject method of treatment, refers to an amount of, e.g., ahedgehog polypeptide in a preparation which, when applied as part of adesired dosage regimen brings about a change in the rate of cellproliferation and/or the state of differentiation of a cell so as toproduce (or inhibit as the case may be) proliferation of lung cells inan amount according to clinically acceptable standards for the disorderto be treated or the cosmetic purpose.

The “growth state” of a cell refers to the rate of proliferation of thecell and the state of differentiation of the cell.

“Homology” and “identity” each refer to sequence similarity between twopolypeptide sequences, with identity being a more strict comparison.Homology and identity can each be determined by comparing a position ineach sequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same amino acidresidue, then the polypeptides can be referred to as identical at thatposition; when the equivalent site is occupied by the same amino acid(e.g., identical) or a similar amino acid (e.g., similar in stericand/or electronic nature), then the molecules can be referred to ashomologous at that position. A percentage of homology or identitybetween sequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40 percent identity, though preferably lessthan 25 percent identity, with a hedgehog sequence disclosed herein.

The term “corresponds to”, when referring to a particular polypeptide ornucleic acid sequence is meant to indicate that the sequence of interestis identical or homologous to the reference sequence to which it is saidto correspond.

The terms “recombinant protein”, “heterologous protein” and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression construct which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a hedgehog polypeptide with a second amino acidsequence defining a domain foreign to and not substantially homologouswith any domain of hh protein. A chimeric protein may present a foreigndomain which is found (albeit in a different protein) in an organismwhich also expresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula (X)_(n)-(hh)_(m)-(Y)_(n), wherein hh represents allor a portion of the hedgehog protein, X and Y each independentlyrepresent an amino acid sequences which are not naturally found as apolypeptide chain contiguous with the hedgehog sequence, m is an integergreater than or equal to 1, and each occurrence of n is, independently,0 or an integer greater than or equal to 1 (n and m are preferably nogreater than 5 or 10).

III. Exemplary Applications of Method and Compositions

The subject method has wide applicability to the treatment orprophylaxis of disorders afflicting lung tissue, as well as in in vitrocultures. In general, the method can be characterized as including astep of administering to an animal an amount of a ptc, hedgehog orfgf-10 therapeutic effective to alter the growth state of a treated lungtissue. The mode of administration and dosage regimens will varydepending on the phenotype of, and desired effect on the target lungtissue. Likewise, as described in further detail below, the use of aparticular ptc, hedgehog or fgf-10 therapeutic, e.g., an agonist orantagonist, will depend on whether proliferation of cells in the treatedlung tissue is desired or intended to be prevented.

In one aspect, the present invention provides pharmaceuticalpreparations and methods for controlling the proliferation of lungtissue utilizing, as an active ingredient, a hedgehog polypeptide or amimetic thereof. The invention also relates to methods of controllingproliferation of mesenchymal and epithelial cells of the tissue by useof the pharmaceutical preparations of the invention.

The formulations of the present invention may be used as part ofregimens in the treatment of disorders of, surgical repair of, ortransplantation of lung tissues and whole organs. The methods andcompositions disclosed herein also provide for the treatment of avariety of proliferative cancerous disorders effecting lung tissue. Forinstance, the subject method can be used to control wound healingprocesses, as for example may be desirable in connection with anysurgery involving lung tissue.

In certain embodiments, the subject compositions can be used to inhibit,rather than promote, growth of lung-derived tissue. For instance,certain of the compositions disclosed herein may be applied to thetreatment or prevention of a variety hyperplastic or neoplasticconditions. The method can find application for the treatment orprophylaxis of, e.g., used to inhibit the growth and metastasis of lungcancer cells. For instance, inhibitory forms of the subject ptc,hedgehog and fgf-10 therapeutics may be used as part of a treatmentprogram for small cell lung cancer (SCLC), as well as non-small celllung cancer (NSCLC), such as adenocarcinoma, lung cell carcinoma andsquamous cell carcinoma.

In other embodiments, the subject method can be used to treat rheumatoidlung disease, which may be marked by pleural thickening, adhesions, andpleural effusions. Such lung (pulmonary) manifestations can occur inboth adult and juvenile forms of rheumatoid arthritis.

In other embodiments, the subject method can be used to treat, or lessenthe severity of, damage to lung tissue as a complication of respiratorydiseases such as broncho-pneumonia, chronic bronchitis, cystic fibrosisand asthma, and bronchospasm, or other apical interstitial lungdiseases, such as cystic fibrosis, ankylosing spondylitis, sarcoidosis,silicosis, eosinophlic granuloma, tuberculosis, and lung infections.

In certain embodiments, the subject method can be used to treat orprevent damage to lung tissue resulting from allergic rhinitis, asthma,emphysema, chronic bronchitis, pneumoconiosis, respiratory distresssyndrome, idiopathic pulmonary fibrosis and primary pulmonaryhypertension

The subject method can be used in the treatment or prevention ofoccupational lung disease such as asbestos-related diseases, silicosis,occupational asthma, coal worker's pneumoconiosis, berylliosis, andindustrial bronchitis.

In still other embodiments, the subject method can be used to treatcertain health consequences of smoking which may result in degenerationof lung tissue.

The subject hedgehog treatments are effective on both human and animalsubjects afflicted with these conditions. Animal subjects to which theinvention is applicable extend to both domestic animals and livestock,raised either as pets or for commercial purposes. Examples are dogs,cats, cattle, horses, sheep, hogs and goats.

Still another aspect of the present invention provides a method ofstimulating the growth and regulating the differentiation of epithelialtissue in tissue culture.

In one embodiment, the subject method can be used to regulate theproliferation and/or differentiation of lung mesenchymal progenitorcells.

The maintenance of lung tissues and whole organs ex vivo is also highlydesirable. Lung and heart-lung transplantation therapy is wellestablished in the treatment of certain human disease. The subjectmethod can be used to maintain the tissue structure of lung tissue exvivo, and in certain embodiments to accelerate the growth of certainlung tissue in vitro.

The present method can also be used for improving the “take rate” of alung transplants in vivo.

IV. Exemplary Hedgehog Therapeutic Compounds

The hedgehog therapeutic compositions of the subject method can begenerated by any of a variety of techniques, including purification ofnaturally occurring proteins, recombinantly produced proteins andsynthetic chemistry. Polypeptide forms of the hedgehog therapeutics arepreferably derived from vertebrate hedgehog proteins, e.g., havesequences corresponding to naturally occurring hedgehog proteins, orfragments thereof, from vertebrate organisms. However, it will beappreciated that the hedgehog polypeptide can correspond to a hedgehogprotein (or fragment thereof) which occurs in any metazoan organism.

The various naturally-occurring hedgehog proteins from which the subjecttherapeutics can be derived are characterized by a signal peptide, ahighly conserved N-terminal region, and a more divergent C-terminaldomain. In addition to signal sequence cleavage in the secretory pathway(Lee, J. J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) GenesDev. 2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),hedgehog precursor proteins naturally undergo an internalautoproteolytic cleavage which depends on conserved sequences in theC-terminal portion (Lee et al. (1994) Science 266:1528-1537; Porter etal. (1995) Nature 374:363-366). This autocleavage leads to a 19 kDN-terminal peptide and a C-terminal peptide of 26-28 kD (Lee et al.(1992) supra; Tabata et al. (1992) supra; Chang et al. (1994) supra; Leeet al. (1994) supra; Bumcrot, D. A., et al. (1995) Mol. Cell. Biol.15:2294-2303; Porter et al. (1995) supra; Ekker, S. C. et al. (1995)Curr. Biol. 5:944-955; Lai, C. J. et al. (1995) Development121:2349-2360). The N-terminal peptide stays tightly associated with thesurface of cells in which it was synthesized, while the C-terminalpeptide is freely diffusible both in vitro and in vivo (Lee et al.(1994) supra; Bumcrot et al. (1995) supra; Mart', E. et al. (1995)Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455).Cell surface retention of the N-terminal peptide is dependent onautocleavage, as a truncated form of hedgehog encoded by an RNA whichterminates precisely at the normal position of internal cleavage isdiffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter, J.A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown thatthe autoproteolytic cleavage of the hedgehog precursor protein proceedsthrough an internal thioester intermediate which subsequently is cleavedin a nucleophilic substitution. It is suggested that the nucleophile isa small lipophilic molecule, more particularly cholesterol, whichbecomes covalently bound to the C-terminal end of the N-peptide (Porteret al. (1996) supra), tethering it to the cell surface.

The vertebrate family of hedgehog genes includes at least four members,e.g., paralogs of the single drosophila hedgehog gene (SEQ ID No. 19).Three of these members, herein referred to as Desert hedgehog (Dhh),Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in allvertebrates, including fish, birds, and mammals. A fourth member, hereinreferred to as tiggie-winkle hedgehog (Thh), appears specific to fish.According to the appended sequence listing, (see also Table 1) a chickenShh polypeptide is encoded by SEQ ID NO:1; a mouse Dhh polypeptide isencoded by SEQ ID No:2; a mouse Ihh polypeptide is encoded by SEQ IDNo:3; a mouse Shh polypeptide is encoded by SEQ ID No:4 a zebrafish Shhpolypeptide is encoded by SEQ ID No:5; a human Shh polypeptide isencoded by SEQ ID No:6; a human Ihh polypeptide is encoded by SEQ IDNo:7; a human Dhh polypeptide is encoded by SEQ ID No. 8; and azebrafish Thh is encoded by SEQ ID No. 9.

TABLE 1 Guide to hedgehog sequences in Sequence Listing Nucleotide AminoAcid Chicken Shh SEQ ID No. 1 SEQ ID No. 10 Mouse Dhh SEQ ID No. 2 SEQID No. 11 Mouse Ihh SEQ ID No. 3 SEQ ID No. 12 Mouse Shh SEQ ID No. 4SEQ ID No. 13 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 14 Human Shh SEQ IDNo. 6 SEQ ID No. 15 Human Ihh SEQ ID No. 7 SEQ ID No. 16 Human Dhh SEQID No. 8 SEQ ID No. 17 Zebrafish Thh SEQ ID No. 9 SEQ ID No. 18Drosophila HH SEQ ID No. 19 SEQ ID No. 20

In addition to the sequence variation between the various hedgehoghomologs, the hedgehog proteins are apparently present naturally in anumber of different forms, including a pro-form, a full-length matureform, and several processed fragments thereof. The pro-form includes anN-terminal signal peptide for directed secretion of the extracellulardomain, while the full-length mature form lacks this signal sequence.

As described above, further processing of the mature form occurs in someinstances to yield biologically active fragments of the protein. Forinstance, sonic hedgehog undergoes additional proteolytic processing toyield two peptides of approximately 19 kDa and 27 kDa, the 19 kDafragment corresponding to an proteolytic N-terminal portion of themature protein.

In addition to proteolytic fragmentation, the vertebrate hedgehogproteins can also be modified post-translationally, such as byglycosylation and/or addition of lipophilic moieties, such as stents,fatty acids, etc., though bacterially produced (e.g. unmodified) formsof the proteins still maintain certain of the bioactivities of thenative protein. Bioactive fragments of hedgehog polypeptides of thepresent invention have been generated and are described in great detailin, e.g., PCT publications WO 95/18856 and WO 96/17924.

There are a wide range of lipophilic moieties with which hedgehogpolypeptides can be derivatived. The term “lipophilic group”, in thecontext of being attached to a hedgehog polypeptide, refers to a grouphaving high hydrocarbon content thereby giving the group high affinityto lipid phases. A lipophilic group can be, for example, a relativelylong chain alkyl or cycloalkyl (preferably n-alkyl) group havingapproximately 7 to 30 carbons. The alkyl group may terminate with ahydroxy or primary amine “tail”. To further illustrate, lipophilicmolecules include naturally-occurring and synthetic aromatic andnon-aromatic moieties such as fatty acids, sterols, esters and alcohols,other lipid molecules, cage structures such as adamantane andbuckminsterfullerenes, and aromatic hydrocarbons such as benzene,perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, andnaphthacene.

In one embodiment, the hedgehog polypeptide is modified with one or moresterol moieties, such as cholesterol. See, for example, PCT publicationWO 96/17924. In certain embodiments, the cholesterol is preferably addedto the C-terminal glycine were the hedgehog polypeptide corresponds tothe naturally-occurring N-terminal proteolytic fragment.

In another embodiment, the hedgehog polypeptide can be modified with afatty acid moiety, such as a myrostoyl, palmitoyl, stearoyl, orarachidoyl moiety. See, e.g., Pepinsky et al. (1998) J. Biol. Chem. 273:14037.

In addition to those effects seen by cholesterol-addition to theC-terminus or fatty acid addition to the N-terminus of extracellularfragments of the protein, at least certain of the biological activitiesof the hedgehog gene products can potentiated by derivativation of theprotein with lipophilic moieties at other sites on the protein and/or bymoieties other than cholesterol or fatty acids. Certain aspects of theinvention are directed to the use of preparations of hedgehogpolypeptides which are modified at sites other than N-terminal orC-terminal residues of the natural processed form of the protein, and/orwhich are modified at such terminal residues with lipophilic moietiesother than a sterol at the C-terminus or fatty acid at the N-terminus.

Particularly useful as lipophilic molecules are alicyclic hydrocarbons,saturated and unsaturated fatty acids and other lipid and phospholipidmoieties, waxes, cholesterol, isoprenoids, terpenes and polyalicyclichydrocarbons including adamantane and buckminsterfullerenes, vitamins,polyethylene glycol or oligoethylene glycol, (C1-C18)-alkyl phosphatediesters, —O—CH2-CH(OH)—O—(C12-C18)-alkyl, and in particular conjugateswith pyrene derivatives. The lipophilic moiety can be a lipophilic dyesuitable for use in the invention include, but are not limited to,diphenylhexatriene, Nile Red, N-phenyl-1-naphthylamine, Prodan,Laurodan, Pyrene, Perylene, rhodamine, rhodamine B,tetramethylrhodamine, Texas Red, sulforhodamine,1,1′-didodecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate,octadecyl rhodamine B and the BODIPY dyes available from MolecularProbes Inc.

Other exemplary lipophilic moieties include aliphatic carbonyl radicalgroups include 1- or 2-adamantylacetyl, 3-methyladamant-1-ylacetyl,3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl,camphaneacetyl, noradamantylacetyl, norbornaneacetyl,bicyclo[2.2.2.]-oct-5-eneacetyl,1-methoxybicyclo[2.2.2]-oct-5-ene-2-carbonyl,cis-5-norbornene-endo-2,3-dicarbonyl, 5-norbornen-2-ylacetyl,(1R)-(−)-myrtentaneacetyl, 2-norbornaneacetyl,anti-3-oxo-tricyclo[2.2.1.0<2,6>]-heptane-7-carbonyl, decanoyl,dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or dodecynoyl.

The hedgehog polypeptide can be linked to the hydrophobic moiety in anumber of ways including by chemical coupling means, or by geneticengineering.

Moreover, mutagenesis can be used to create modified hh polypeptides,e.g., for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides can beproduced, for instance, by amino acid substitution, deletion, oraddition. Modified hedgehog polypeptides can also include those withaltered post-translational processing relative to a naturally occurringhedgehog protein, e.g., altered glycosylation, cholesterolization,prenylation and the like.

In one embodiment, the hedgehog therapeutic is a polypeptide encodableby a nucleotide sequence that hybridizes under stringent conditions to ahedgehog coding sequence represented in one or more of SEQ ID Nos:1-7.Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C.

As described in the literature, genes for other hedgehog proteins, e.g.,from other animals, can be obtained from mRNA or genomic DNA samplesusing techniques well known in the art. For example, a cDNA encoding ahedgehog protein can be obtained by isolating total mRNA from a cell,e.g. a mammalian cell, e.g. a human cell, including embryonic cells.Double stranded cDNAs can then be prepared from the total mRNA, andsubsequently inserted into a suitable plasmid or bacteriophage vectorusing any one of a number of known techniques. The gene encoding ahedgehog protein can also be cloned using established polymerase chainreaction techniques.

Preferred nucleic acids encode a hedgehog polypeptide comprising anamino acid sequence at least 60% homologous or identical, morepreferably 70% homologous or identical, and most preferably 80%homologous or identical with an amino acid sequence selected from thegroup consisting of SEQ ID Nos:8-14. Nucleic acids which encodepolypeptides at least about 90%, more preferably at least about 95%, andmost preferably at least about 98-99% homology or identity with an aminoacid sequence represented in one of SEQ ID Nos:8-14 are also within thescope of the invention.

In addition to native hedgehog proteins, hedgehog polypeptides preferredby the present invention are at least 60% homologous or identical, morepreferably 70% homologous or identical and most preferably 80%homologous or identical with an amino acid sequence represented by anyof SEQ ID Nos:8-14. Polypeptides which are at least 90%, more preferablyat least 95%, and most preferably at least about 98-99% homologous oridentical with a sequence selected from the group consisting of SEQ IDNos:8-14 are also within the scope of the invention. The onlyprerequisite is that the hedgehog polypeptide is capable of modulatingthe growth of lung cells.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a hedgehog polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant hedgehog gene, is meant to includewithin the meaning of “recombinant protein” those proteins having anamino acid sequence of a native hedgehog protein, or an amino acidsequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the protein.

The method of the present invention can also be carried out usingvariant forms of the naturally occurring hedgehog polypeptides, e.g.,mutational variants.

As is known in the art, hedgehog polypeptides can be produced bystandard biological techniques or by chemical synthesis. For example, ahost cell transfected with a nucleic acid vector directing expression ofa nucleotide sequence encoding the subject polypeptides can be culturedunder appropriate conditions to allow expression of the peptide tooccur. The polypeptide hedgehog may be secreted and isolated from amixture of cells and medium containing the recombinant hedgehogpolypeptide. Alternatively, the peptide may be retained cytoplasmicallyby removing the signal peptide sequence from the recombinant hedgehoggene and the cells harvested, lysed and the protein isolated. A cellculture includes host cells, media and other byproducts. Suitable mediafor cell culture are well known in the art. The recombinant hedgehogpolypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant hedgehog polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as an hedgehog/GST fusionprotein. The host cell may be any prokaryotic or eukaryotic cell.

Recombinant hedgehog genes can be produced by ligating nucleic acidencoding an hedgehog protein, or a portion thereof, into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject hedgehog polypeptides include plasmids and other vectors. Forinstance, suitable vectors for the expression of a hedgehog polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due to the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an hedgehog polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the hedgehog genes represented in SEQ IDNos:1-7.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinanthedgehog polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

When it is desirable to express only a portion of an hedgehog protein,such as a form lacking a portion of the N-terminus, i.e. a truncationmutant which lacks the signal peptide, it may be necessary to add astart codon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing hedgehog-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. It is widely appreciated that fusionproteins can also facilitate the expression of proteins, andaccordingly, can be used in the expression of the hedgehog polypeptidesof the present invention. For example, hedgehog polypeptides can begenerated as glutathione-S-transferase (GST-fusion) proteins. SuchGST-fusion proteins can enable easy purification of the hedgehogpolypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)). In another embodiment,a fusion gene coding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence, can be used to replacethe signal sequence which naturally occurs at the N-terminus of thehedgehog protein (e.g. of the pro-form, in order to permit purificationof the poly(His)-hedgehog protein by affinity chromatography using aNi²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase (e.g., see Hochuliet al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Hedgehog polypeptides may also be chemically modified to create hedgehogderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, cholesterol, isoprenoids,lipids, phosphate, acetyl groups and the like. Covalent derivatives ofhedgehog proteins can be prepared by linking the chemical moieties tofunctional groups on amino acid sidechains of the protein or at theN-terminus or at the C-terminus of the polypeptide.

For instance, hedgehog proteins can be generated to include a moiety,other than sequence naturally associated with the protein, that binds acomponent of the extracellular matrix and enhances localization of theanalog to cell surfaces. For example, sequences derived from thefibronectin “type-III repeat”, such as a tetrapeptide sequence R-G-D-S(Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt et al.(1985) EMBO 4:1755-9) can be added to the hedgehog polypeptide tosupport attachment of the chimeric molecule to a cell through bindingECM components (Ruoslahti et al. (1987) Science 238:491-497;Pierschbacher et al. (1987) J. Biol. Chem. 262:17294-8.; Hynes (1987)Cell 48:549-54; and Hynes (1992) Cell 69:11-25).

In a preferred embodiment, the hedgehog polypeptide is isolated from, oris otherwise substantially free of, other cellular proteins, especiallyother extracellular or cell surface associated proteins which maynormally be associated with the hedgehog polypeptide, unless provided inthe form of fusion protein with the hedgehog polypeptide. The term“substantially free of other cellular or extracellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially purepreparations” or “purified preparations” are defined as encompassingpreparations of hedgehog polypeptides having less than 20% (by dryweight) contaminating protein, and preferably having less than 5%contaminating protein. By “purified”, it is meant that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules, such as other proteins. The term “purified” as usedherein preferably means at least 80% by dry weight, more preferably inthe range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above.

As described above for recombinant polypeptides, isolated hedgehogpolypeptides can include all or a portion of the amino acid sequencesrepresented in any of SEQ ID Nos:10-18 or 20, or a homologous sequencethereto. Preferred fragments of the subject hedgehog proteins correspondto the N-terminal and C-terminal proteolytic fragments of the matureprotein. Bioactive fragments of hedgehog polypeptides are described ingreat detail in PCT publications WO 95/18856 and WO 96/17924.

With respect to bioactive fragments of hedgehog polypeptide, preferredhedgehog therapeutics include at least 50 (contiguous) amino acidresidues of a hedgehog polypeptide, more preferably at least 100(contiguous), and even more preferably at least 150 (contiguous)residues.

Another preferred hedgehog polypeptide which can be included in thehedgehog therapeutic is an N-terminal fragment of the mature proteinhaving a molecular weight of approximately 19 kDa.

Preferred human hedgehog proteins include N-terminal fragmentscorresponding approximately to residues 24-197 of SEQ ID No. 15, 28-202of SEQ ID No. 16, and 23-198 of SEQ ID No. 17. By “correspondingapproximately” it is meant that the sequence of interest is at most 20amino acid residues different in length to the reference sequence,though more preferably at most 5, 10 or 15 amino acid different inlength.

As described above for recombinant polypeptides, isolated hedgehogpolypeptides can include all or a portion of the amino acid sequencesrepresented in SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQID No:12, SEQ ID No:13 or SEQ ID No:14, or a homologous sequencethereto. Preferred fragments of the subject hedgehog proteins correspondto the N-terminal and C-terminal proteolytic fragments of the matureprotein. Bioactive fragments of hedgehog polypeptides are described ingreat detail in PCT publications WO 95/18856 and WO 96/17924.

Still other preferred hedgehog polypeptides includes an amino acidsequence represented by the formula A-B wherein: (i) A represents all orthe portion of the amino acid sequence designated by residues 1-168 ofSEQ ID No:21; and B represents at least one amino acid residue of theamino acid sequence designated by residues 169-221 of SEQ ID No:21; (ii)A represents all or the portion of the amino acid sequence designated byresidues 24-193 of SEQ ID No:15; and B represents at least one aminoacid residue of the amino acid sequence designated by residues 194-250of SEQ ID No:15; (iii) A represents all or the portion of the amino acidsequence designated by residues 25-193 of SEQ ID No:13; and B representsat least one amino acid residue of the amino acid sequence designated byresidues 194-250 of SEQ ID No:13; (iv) A represents all or the portionof the amino acid sequence designated by residues 23-193 of SEQ IDNo:11; and B represents at least one amino acid residue of the aminoacid sequence designated by residues 194-250 of SEQ ID No:11; (v) Arepresents all or the portion of the amino acid sequence designated byresidues 28-197 of SEQ ID No:12; and B represents at least one aminoacid residue of the amino acid sequence designated by residues 198-250of SEQ ID No:12; (vi) A represents all or the portion of the amino acidsequence designated by residues 29-197 of SEQ ID No:16; and B representsat least one amino acid residue of the amino acid sequence designated byresidues 198-250 of SEQ ID No:16; or (vii) A represents all or theportion of the amino acid sequence designated by residues 23-193 of SEQID No. 17, and B represents at least one amino acid residue of the aminoacid sequence designated by residues 194-250 of SEQ ID No. 17. Incertain preferred embodiments, A and B together represent a contiguouspolypeptide sequence designated sequence, A represents at least 25, 50,75, 100, 125 or 150 (contiguous) amino acids of the designated sequence,and B represents at least 5, 10, or 20 (contiguous) amino acid residuesof the amino acid sequence designated by corresponding entry in thesequence listing, and A and B together preferably represent a contiguoussequence corresponding to the sequence listing entry. Similar fragmentsfrom other hedgehog also contemplated, e.g., fragments which correspondto the preferred fragments from the sequence listing entries which areenumerated above. In preferred embodiments, the hedgehog polypeptideincludes a C-terminal glycine (or other appropriate residue) which isderivatized with a cholesterol.

Isolated peptidyl portions of hedgehog proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a hedgehog polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) hedgehog protein. For example, Romanet al. (1994) Eur J Biochem 222:65-73 describe the use ofcompetitive-binding assays using short, overlapping synthetic peptidesfrom larger proteins to identify binding domains.

The recombinant hedgehog polypeptides of the present invention alsoinclude homologs of the authentic hedgehog proteins, such as versions ofthose protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter potential cleavage sequences orwhich inactivate an enzymatic activity associated with the protein.Hedgehog homologs of the present invention also include proteins whichhave been post-translationally modified in a manner different than theauthentic protein. Exemplary derivatives of hedgehog proteins includepolypeptides which lack N-glycosylation sites (e.g. to produce anunglycosylated protein), which lack sites for cholesterolization, and/orwhich lack N-terminal and/or C-terminal sequences.

Modification of the structure of the subject hedgehog polypeptides canalso be for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides, when designedto retain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the hedgehogpolypeptides described in more detail herein. Such modified peptides canbe produced, for instance, by amino acid substitution, deletion, oraddition.

It is well known in the art that one could reasonably expect thatcertain isolated replacements of amino acids, e.g., replacement of anamino acid residue with another related amino acid (i.e. isostericand/or isoelectric mutations), can be carried out without major effecton the biological activity of the resulting molecule. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare can be divided into four families: (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer,WH Freeman and Co.: 1981). Whether a change in the amino acid sequenceof a peptide results in a functional hedgehog homolog (e.g. functionalin the sense that it acts to mimic or antagonize the wild-type form) canbe readily determined by assessing the ability of the variant peptide toproduce a response in cells in a fashion similar to the wild-typeprotein, or competitively inhibit such a response. Polypeptides in whichmore than one replacement has taken place can readily be tested in thesame manner.

It is specifically contemplated that the methods of the presentinvention can be carried using homologs of naturally occurring hedgehogproteins. In one embodiment, the invention contemplates using hedgehogpolypeptides generated by combinatorial mutagenesis. Such methods, asare known in the art, are convenient for generating both point andtruncation mutants, and can be especially useful for identifyingpotential variant sequences (e.g. homologs) that are functional inbinding to a receptor for hedgehog proteins. The purpose of screeningsuch combinatorial libraries is to generate, for example, novel hedgehoghomologs which can act as either agonists or antagonist. To illustrate,hedgehog homologs can be engineered by the present method to providemore efficient binding to a cognate receptor, such as patched, yet stillretain at least a portion of an activity associated with hedgehog. Thus,combinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein. Likewise,hedgehog homologs can be generated by the present combinatorial approachto act as antagonists, in that they are able to mimic, for example,binding to other extracellular matrix components (such as receptors),yet not induce any biological response, thereby inhibiting the action ofauthentic hedgehog or hedgehog agonists. Moreover, manipulation ofcertain domains of hedgehog by the present method can provide domainsmore suitable for use in fusion proteins, such as one that incorporatesportions of other proteins which are derived from the extracellularmatrix and/or which bind extracellular matrix components.

To further illustrate the state of the art of combinatorial mutagenesis,it is noted that the review article of Gallop et al. (1994) J Med Chem37:1233 describes the general state of the art of combinatoriallibraries as of the earlier 1990's. In particular, Gallop et al state atpage 1239 “[s]creening the analog libraries aids in determining theminimum size of the active sequence and in identifying those residuescritical for binding and intolerant of substitution”. In addition, theLadner et al. PCT publication WO90/02809, the Goeddel et al. U.S. Pat.No. 5,223,408, and the Markland et al. PCT publication WO92/15679illustrate specific techniques which one skilled in the art couldutilize to generate libraries of hedgehog variants which can be rapidlyscreened to identify variants/fragments which retained a particularactivity of the hedgehog polypeptides. These techniques are exemplary ofthe art and demonstrate that large libraries of relatedvariants/truncants can be generated and assayed to isolate particularvariants without undue experimentation. Gustin et al. (1993) Virology193:653, and Bass et al. (1990) Proteins: Structure, Function andGenetics 8:309-314

also describe other exemplary techniques from the art which can beadapted as means for generating mutagenic variants of hedgehogpolypeptides.

Indeed, it is plain from the combinatorial mutagenesis art that largescale mutagenesis of hedgehog proteins, without any preconceived ideasof which residues were critical to the biological function, and generatewide arrays of variants having equivalent biological activity. Indeed,it is the ability of combinatorial techniques to screen billions ofdifferent variants by high throughout analysis that removes anyrequirement of a priori understanding or knowledge of critical residues.

To illustrate, the amino acid sequences for a population of hedgehoghomologs or other related proteins are aligned, preferably to promotethe highest homology possible. Such a population of variants caninclude, for example, hedgehog homologs from one or more species. Aminoacids which appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences. In apreferred embodiment, the variegated library of hedgehog variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential hedgehog sequencesare expressible as individual polypeptides, or alternatively, as a setof larger fusion proteins (e.g. for phage display) containing the set ofhedgehog sequences therein.

As illustrated in PCT publication WO 95/18856, to analyze the sequencesof a population of variants, the amino acid sequences of interest can bealigned relative to sequence homology. The presence or absence of aminoacids from an aligned sequence of a particular variant is relative to achosen consensus length of a reference sequence, which can be real orartificial.

In an illustrative embodiment, alignment of exons 1, 2 and a portion ofexon 3 encoded sequences (e.g. the N-terminal approximately 221 residuesof the mature protein) of each of the Shh clones produces a degenerateset of Shh polypeptides represented by the general formula:

(SEQ ID No: 21 C-G-P-G-R-G-X(1)-G-X(2)-R-R-H-P-K-K-L-T-P-L-A-Y-K-Q-F-I-P-N-V-A-E-K-T-L-G-A-S-G-R-Y-E-G-K-I-X(3)-R-N-S-E-R-F-K-E-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-T-G-A-D-R-L-M-T-Q-R-C-K-D-K-L-N-X(4)-L-A-I-S-V-M-N-X(5)-W-P-G-V-X(6)-L-R-V-T-E-G-W-D-E-D-G-H-H-X(7)-E-E-S-L-H-Y-E-G-R-A-V-D-I-T-T-S-D-R-D-X(8)-S-K-Y-G-X(9)-L-X(10)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-K-A-H-I-H-C-S-V-K-A-E-N-S-V-A-A-K-S-G-G-C-F-P-G-S-A-X(11)-V-X(12)-L-X(13)-X(14)-G-G-X(15)-K-X-(16)-V-K-D-L-X(17)-P-G-D-X(18)-V-L-A-A-D-X(19)-X(20)-G-X(21)-L-X(22)-X(23)-S-D-F-X(24)-X(25)-F-X(26)- D-Rwherein each of the degenerate positions “X” can be an amino acid whichoccurs in that position in one of the human, mouse, chicken or zebrafishShh clones, or, to expand the library, each X can also be selected fromamongst amino acid residue which would be conservative substitutions forthe amino acids which appear naturally in each of those positions. Forinstance, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Phe, Tyr or Trp;Xaa(2) represents Arg, His or Lys; Xaa(3) represents Gly, Ala, Val, Leu,Ile, Ser or Thr; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;Xaa(5) represents Lys, Arg, His, Asn or Gln; Xaa(6) represents Lys, Argor His; Xaa(7) represents Ser, Thr, Tyr, Trp or Phe; Xaa(8) representsLys, Arg or His; Xaa(9) represents Met, Cys, Ser or Thr; Xaa(10)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11) represents Leu,Val, Met, Thr or Ser; Xaa(12) represents His, Phe, Tyr, Ser, Thr, Met orCys; Xaa(13) represents Gln, Asn, Glu, or Asp; Xaa(14) represents His,Phe, Tyr, Thr, Gln, Asn, Glu or Asp; Xaa(15) represents Gln, Asn, Glu,Asp, Thr, Ser, Met or Cys; Xaa(16) represents Ala, Gly, Cys, Leu, Val orMet; Xaa(17) represents Arg, Lys, Met, Ile, Asn, Asp, Glu, Gln, Ser, Thror Cys; Xaa(18) represents Arg, Lys, Met or Ile; Xaa(19) represents Ala,Gly, Cys, Asp, Glu, Gln, Asn, Ser, Thr or Met; Xaa(20) represents Ala,Gly, Cys, Asp, Asn, Glu or Gln; Xaa(21) represents Arg, Lys, Met, Ile,Asn, Asp, Glu or Gln; Xaa(22) represent Leu, Val, Met or Ile; Xaa(23)represents Phe, Tyr, Thr, His or Trp; Xaa(24) represents Ile, Val, Leuor Met; Xaa(25) represents Met, Cys, Ile, Leu, Val, Thr or Ser; Xaa(26)represents Leu, Val, Met, Thr or Ser. In an even more expansive library,each X can be selected from any amino acid.

In similar fashion, alignment of each of the human, mouse, chicken andzebrafish hedgehog clones, can provide a degenerate polypeptide sequencerepresented by the general formula:

(SEQ ID No: 22 C-G-P-G-R-G-X(1)-X(2)-X(3)-R-R-X(4)-X(5)-X(6)-P-K-X(7)-L-X(8)-P-L-X(9)-Y-K-Q-F-X(10)-P-X(11)-X(12)-X(13)-E-X(14)-T-L-G-A-S-G-X(15)-X(16)-E-G-X(17)-X(18)-X(19)-R-X(20)-S-E-R-F-X(21)-X(22)-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-X(23)-G-A-D-R-L-M-T-X(24)-R-C-K-X(25)-X(26)-X(27)-N-X(28)-L-A-I-S-V-M-N-X(29)-W-P-G-V-X(30)-L-R-V-T-E-G-X(31)-D-E-D-G-H-H-X(32)-X(33)-X(34)-S-L-H-Y-E-G-R-A-X(35)-D-I-T-T-S-D-R-D-X(36)-X(37)-K-Y-G-X(38)-L-X(39)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-X(40)-X(41)-H-X(42)-H-X(43)-S-V-K-X(44)-X(45)wherein, as above, each of the degenerate positions “X” can be an aminoacid which occurs in a corresponding position in one of the wild-typeclones, and may also include amino acid residue which would beconservative substitutions, or each X can be any amino acid residue. Inan exemplary embodiment, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Pro,Phe or Tyr; Xaa(2) represents Gly, Ala, Val, Leu or Ile; Xaa(3)represents Gly, Ala, Val, Leu, Ile, Lys, His or Arg; Xaa(4) representsLys, Arg or His; Xaa(5) represents Phe, Trp, Tyr or an amino acid gap;Xaa(6) represents Gly, Ala, Val, Leu, Ile or an amino acid gap; Xaa(7)represents Asn, Gln, His, Arg or Lys; Xaa(8) represents Gly, Ala, Val,Leu, Ile, Ser or Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser orThr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11)represents Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala,Val, Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile orPro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly, Ala,Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly, Ala, Val,Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys; Xaa(18)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19) represents Thror Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile, Asn or Gln; Xaa(21)represents Arg, His or Lys; Xaa(22) represents Asp or Glu; Xaa(23)represents Ser or Thr; Xaa(24) represents Glu, Asp, Gln or Asn; Xaa(25)represents Glu or Asp; Xaa(26) represents Arg, His or Lys; Xaa(27)represents Gly, Ala, Val, Leu or Ile; Xaa(28) represents Gly, Ala, Val,Leu, Ile, Thr or Ser; Xaa(29) represents Met, Cys, Gln, Asn, Arg, Lys orHis; Xaa(30) represents Arg, His or Lys; Xaa(31) represents Trp, Phe,Tyr, Arg, His or Lys; Xaa(32) represents Gly, Ala, Val, Leu, Ile, Ser,Thr, Tyr or Phe; Xaa(33) represents Gln, Asn, Asp or Glu; Xaa(34)represents Asp or Glu; Xaa(35) represents Gly, Ala, Val, Leu, or Ile;Xaa(36) represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr orSer; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or Cys;Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(40)represents Arg, His or Lys; Xaa(41) represents Asn, Gln, Gly, Ala, Val,Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or Ile; Xaa(43)represents Gly, Ala, Val, Leu, Ile, Ser, Thr or Cys; Xaa(44) representsGly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45) represents Asp or Glu.

There are many ways by which the library of potential hedgehog homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential hedgehog sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis of hedgehoghomologs. The most widely used techniques for screening large genelibraries typically comprises cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the illustrative assays described below areamenable to high through-put analysis as necessary to screen largenumbers of degenerate hedgehog sequences created by combinatorialmutagenesis techniques.

In one embodiment, the combinatorial library is designed to be secreted(e.g. the polypeptides of the library all include a signal sequence butno transmembrane or cytoplasmic domains), and is used to transfect aeukaryotic cell that can be co-cultured with lung cells, e.g., lungmesenchymal or epithelial cells. A functional hedgehog protein secretedby the cells expressing the combinatorial library will diffuse to theneighboring lung cells and induce a particular biological response, suchas proliferation. The pattern of detection of proliferation willresemble a gradient function, and will allow the isolation (generallyafter several repetitive rounds of selection) of cells producinghedgehog homologs active as proliferative agents with respect to thelung cells. Likewise, hedgehog antagonists can be selected in similarfashion by the ability of the cell producing a functional antagonist toprotect neighboring cells (e.g., to inhibit proliferation) from theeffect of wild-type hedgehog added to the culture media.

To illustrate, target lung cells are cultured in 24-well microtitreplates. Other eukaryotic cells are transfected with the combinatorialhedgehog gene library and cultured in cell culture inserts (e.g.Collaborative Biomedical Products, Catalog #40446) that are able to fitinto the wells of the microtitre plate. The cell culture inserts areplaced in the wells such that recombinant hedgehog homologs secreted bythe cells in the insert can diffuse through the porous bottom of theinsert and contact the target cells in the microtitre plate wells. Aftera period of time sufficient for functional forms of a hedgehog proteinto produce a measurable response in the target cells, such asproliferation, the inserts are removed and the effect of the varianthedgehog proteins on the target cells determined. Cells from the insertscorresponding to wells which score positive for activity can be splitand re-cultured on several inserts, the process being repeated until theactive clones are identified.

In yet another screening assay, the candidate hedgehog gene products aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to associate with a hedgehog-bindingmoiety (such as the patched protein or other hedgehog receptor) via thisgene product is detected in a “panning assay”. Such panning steps can becarried out on cells cultured from embryos. For instance, the genelibrary can be cloned into the gene for a surface membrane protein of abacterial cell, and the resulting fusion protein detected by panning(Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similarfashion, fluorescently labeled molecules which bind hedgehog can be usedto score for potentially functional hedgehog homologs. Cells can bevisually inspected and separated under a fluorescence microscope, or,where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and f1 are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffths et al. (1993) EMBO J. 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

In an illustrative embodiment, the recombinant phage antibody system(RPAS, Pharamacia Catalog number 27-9400-01) can be easily modified foruse in expressing and screening hedgehog combinatorial libraries. Forinstance, the pCANTAB 5 phagemid of the RPAS kit contains the gene whichencodes the phage gIII coat protein. The hedgehog combinatorial genelibrary can be cloned into the phagemid adjacent to the gIII signalsequence such that it will be expressed as a gIII fusion protein. Afterligation, the phagemid is used to transform competent E. coli TG1 cells.Transformed cells are subsequently infected with M13KO7 helper phage torescue the phagemid and its candidate hedgehog gene insert. Theresulting recombinant phage contain phagemid DNA encoding a specificcandidate hedgehog, and display one or more copies of the correspondingfusion coat protein. The phage-displayed candidate hedgehog proteinswhich are capable of binding an hedgehog receptor are selected orenriched by panning. For instance, the phage library can be applied tocells which express the patched protein and unbound phage washed awayfrom the cells. The bound phage is then isolated, and if the recombinantphage express at least one copy of the wild type gIII coat protein, theywill retain their ability to infect E. coli. Thus, successive rounds ofreinfection of E. coli, and panning will greatly enrich for hedgehoghomologs, which can then be screened for further biological activitiesin order to differentiate agonists and antagonists.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays such as phage display.To overcome this problem, a new technique has been developed recently,recursive ensemble mutagenesis (REM), which allows one to avoid the veryhigh proportion of non-functional proteins in a random library andsimply enhances the frequency of functional proteins, thus decreasingthe complexity required to achieve a useful sampling of sequence space.REM is an algorithm which enhances the frequency of functional mutantsin a library when an appropriate selection or screening method isemployed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan etal., 1992, Parallel Problem Solving from Nature, 2., In Maenner andManderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410;Delgrave et al., 1993, Protein Engineering 6(3):327-331).

The invention also provides for reduction of the hedgehog protein togenerate mimetics, e.g. peptide or non-peptide agents, which are able todisrupt binding of a hedgehog polypeptide of the present invention withan hedgehog receptor. Thus, such mutagenic techniques as described aboveare also useful to map the determinants of the hedgehog proteins whichparticipate in protein-protein interactions involved in, for example,binding of the subject hedgehog polypeptide to other extracellularmatrix components. To illustrate, the critical residues of a subjecthedgehog polypeptide which are involved in molecular recognition of anhedgehog receptor such as patched can be determined and used to generatehedgehog-derived peptidomimetics which competitively inhibit binding ofthe authentic hedgehog protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of each ofthe subject hedgehog proteins which are involved in binding otherextracellular proteins, peptidomimetic compounds can be generated whichmimic those residues of the hedgehog protein which facilitate theinteraction. Such mimetics may then be used to interfere with the normalfunction of a hedgehog protein. For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

Recombinantly produced forms of the hedgehog proteins can be producedusing, e.g, expression vectors containing a nucleic acid encoding ahedgehog polypeptide, operably linked to at least one transcriptionalregulatory sequence. Operably linked is intended to mean that thenucleotide sequence is linked to a regulatory sequence in a manner whichallows expression of the nucleotide sequence. Regulatory sequences areart-recognized and are selected to direct expression of a hedgehogpolypeptide. Accordingly, the term transcriptional regulatory sequenceincludes promoters, enhancers and other expression control elements.Such regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences, sequences that control the expression of a DNA sequence whenoperatively linked to it, may be used in these vectors to express DNAsequences encoding hedgehog polypeptide. Such useful expression controlsequences, include, for example, a viral LTR, such as the LTR of theMoloney murine leukemia virus, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage λ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other proteins encoded by the vector, such as antibiotic markers,should also be considered.

In addition to providing a ready source of hedgehog polypeptides forpurification, the gene constructs of the present invention can also beused as a part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of a hedgehogpolypeptide. Thus, another aspect of the invention features expressionvectors for in vivo transfection of a hedgehog polypeptide in particularcell types so as cause ectopic expression of a hedgehog polypeptide inlung tissue.

Formulations of such expression constructs may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the recombinant gene to cells in vivo.Approaches include insertion of the hedgehog coding sequence in viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNAcan be delivered with the help of, for example, cationic liposomes(lipofectin) or derivatized (e.g. antibody conjugated), polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular carriers, as well as direct injection of the geneconstruct or CaPO₄ precipitation carried out in vivo. It will beappreciated that because transduction of appropriate target cellsrepresents the critical first step in gene therapy, choice of theparticular gene delivery system will depend on such factors as thephenotype of the intended target and the route of administration, e.g.locally or systemically. Furthermore, it will be recognized that theparticular gene construct provided for in vivo transduction of hedgehogexpression are also useful for in vitro transduction of cells, such asfor use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding the particular form of the hedgehog polypeptide desired.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding ahedgehog polypeptide and renders the retrovirus replication defective.The replication defective retrovirus is then packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include Crip, Cre, 2 andAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including lung cells, in vitro and/or in vivo(see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos andMulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990)Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl.Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci.USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the hedgehog gene of the retroviral vector.

Another viral gene delivery system useful in the present method utilizesadenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they can be used to infect a wide variety of celltypes, including lung cells (Rosenfeld et al. (1992) cited supra).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use and therefore favored by the presentinvention are deleted for all or parts of the viral E1 and E3 genes butretain as much as 80% of the adenoviral genetic material (see, e.g.,Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham etal. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton,N.J., 1991) vol. 7. pp. 109-127). Expression of the inserted hedgehoggene can be under control of, for example, the E1A promoter, the majorlate promoter (MLP) and associated leader sequences, the E3 promoter, orexogenously added promoter sequences.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a hedgehogpolypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the hedgehog polypeptidegene by the targeted cell. Exemplary gene delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutichedgehog gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057). A hedgehog expression construct can bedelivered in a gene therapy construct to dermal cells by, e.g.,electroporation using techniques described, for example, by Dev et al.((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

In yet another embodiment, the ptc, hedgehog or fgf-10 therapeutic canbe a “gene activation” construct which, by homologous recombination witha genomic DNA, alters the transcriptional regulatory sequences of anendogenous gene. For instance, the gene activation construct can replacethe endogenous promoter of a hedgehog gene with a heterologous promoter,e.g., one which causes constitutive expression of the hedgehog gene orwhich causes inducible expression of the gene under conditions differentfrom the normal expression pattern of the gene. Other genes in thepatched signaling pathway can be similarly targeted. A variety ofdifferent formats for the gene activation constructs are available. See,for example, the Transkaryotic Therapies, Inc PCT publicationsWO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.

In preferred embodiments, the nucleotide sequence used as the geneactivation construct can be comprised of (1) DNA from some portion ofthe endogenous hedgehog gene (exon sequence, intron sequence, promotersequences, etc.) which direct recombination and (2) heterologoustranscriptional regulatory sequence(s) which is to be operably linked tothe coding sequence for the genomic hedgehog gene upon recombination ofthe gene activation construct. For use in generating cultures ofhedgehog producing cells, the construct may further include a reportergene to detect the presence of the knockout construct in the cell.

The gene activation construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to provide theheterologous regulatory sequences in operative association with thenative hedgehog gene. Such insertion occurs by homologous recombination,i.e., recombination regions of the activation construct that arehomologous to the endogenous hedgehog gene sequence hybridize to thegenomic DNA and recombine with the genomic sequences so that theconstruct is incorporated into the corresponding position of the genomicDNA.

The terms “recombination region” or “targeting sequence” refer to asegment (i.e., a portion) of a gene activation construct having asequence that is substantially identical to or substantiallycomplementary to a genomic gene sequence, e.g., including 5′ flankingsequences of the genomic gene, and can facilitate homologousrecombination between the genomic sequence and the targeting transgeneconstruct.

As used herein, the term “replacement region” refers to a portion of aactivation construct which becomes integrated into an endogenouschromosomal location following homologous recombination between arecombination region and a genomic sequence.

The heterologous regulatory sequences, e.g., which are provided in thereplacement region, can include one or more of a variety elements,including: promoters (such as constitutive or inducible promoters),enhancers, negative regulatory elements, locus control regions,transcription factor binding sites, or combinations thereof.Promoters/enhancers which may be used to control the expression of thetargeted gene in vivo include, but are not limited to, thecytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.Exp. Med, 169:13), the human β-actin promoter (Gunning et al. (1987)PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal. (1984) Mol. Cell. Biol. 4:1354-1362), the long terminal repeatsequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.(1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell. Biol.,4:817; and Sprague et al. (1983) J, Virol., 45:773), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus(HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et al.(1992) Nature Genetics, 1:379-384).

In an exemplary embodiment, portions of the 5′ flanking region of thehuman Shh gene are amplified using primers which add restriction sites,to generate the following fragments

5′-gcgcgcttcgaaGCGAGGCAGCCAGCGAGGGAGAGAGCGAGCGGGCGAGCCGGAGCGAGGAAatcgatgcgcgc (primer 1)5′-gcgcgcagatctGGGAAAGCGCAAGAGAGAGCGCACACGCACACACCCGCCGCGCGCACTCGggatccgcgcgc (primer 2)As illustrated, primer 1 includes a 5′ non-coding region of the humanShh gene and is flanked by an AsuII and ClaI restriction sites. Primer 2includes a portion of the 5′ non-coding region immediately 3′ to thatpresent in primer 1. The hedgehog gene sequence is flanked by XhoII andBamHI restriction sites. The purified amplimers are cut with each of theenzymes as appropriate.

The vector pcDNA1.1 (Invitrogen) includes a CMV promoter. The plasmid iscut with AsuII, which cleaves just 3′ to the CMV promoter sequence. TheAsuII/ClaI fragment of primer 1 is ligated to the AsuII cleavage site ofthe pcDNA vector. The ClaI/AsuII ligation destroys the AsuII site at the3′ end of a properly inserted primer 1.

The vector is then cut with BamHI, and an XhoII/BamHI fragment of primer2 is ligated to the BamHI cleavage site. As above, the BamHI/XhoIIligation destroys the BamHI site at the 5′ end of a properly insertedprimer 2.

Individual colonies are selected, cut with AsuII and BamHI, and the sizeof the AsuII/BamHI fragment determined. Colonies in which both theprimer 1 and primer 2 sequences are correctly inserted are furtheramplified, an cut with AsuII and BamHI to produce the gene activationconstruct

cgaagcgaggcagccagcgagggagagagcgagcgggcgagccggagcgaggaaATCGAAGGTTCGAATCCTTCCCCCACCACCATCACTTTCAAAAGTCCGAAAGAATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGTAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCgatctgggaaagcgcaagagagagcgcacacgcacacacccgccgcg cgcactcggIn this construct, the flanking primer 1 and primer 2 sequences providethe recombination region which permits the insertion of the CMV promoterin front of the coding sequence for the human Shh gene. Otherheterologous promoters (or other transcriptional regulatory sequences)can be inserted in a genomic hedgehog gene by a similar method.

In still other embodiments, the replacement region merely deletes anegative transcriptional control element of the native gene, e.g., toactivate expression, or ablates a positive control element, e.g., toinhibit expression of the targeted gene.

V. Exemplary Ptc Therapeutic Compounds

In another embodiment, the subject method is carried out using a ptctherapeutic composition. Such compositions can be generated with, forexample, compounds which bind to patched and alter its signaltransduction activity, compounds which alter the binding and/orenzymatic activity of a protein (e.g., intracellular) involved inpatched signal pathway, and compounds which alter the level ofexpression of a hedgehog protein, a patched protein or a proteininvolved in the intracellular signal transduction pathway of patched.

The availability of purified and recombinant hedgehog polypeptidesfacilitates the generation of assay systems which can be used to screenfor drugs, such as small organic molecules, which are either agonists orantagonists of the normal cellular function of a hedgehog and/or patchedprotein, particularly their role in the pathogenesis of proliferationand/or differentiation of various lung cells and maintenance of lungtissue. In one embodiment, the assay evaluates the ability of a compoundto modulate binding between a hedgehog polypeptide and a hedgehogreceptor such as patched. In other embodiments, the assay merely scoresfor the ability of a test compound to alter the signal transductionacitity of the patched protein. In this manner, a variety of hedgehogand/or ptc therapeutics, both proliferative and anti-proliferative inactivity, can be identified. A variety of assay formats will sufficeand, in light of the present disclosure, will be comprehended by skilledartisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with receptorproteins.

Accordingly, in an exemplary screening assay for ptc therapeutics, thecompound of interest is contacted with a mixture including a hedgehogreceptor protein (e.g., a cell expressing the patched receptor) and ahedgehog protein under conditions in which it is ordinarily capable ofbinding the hedgehog protein. To the mixture is then added a compositioncontaining a test compound. Detection and quantification ofreceptor/hedgehog complexes provides a means for determining the testcompound's efficacy at inhibiting (or potentiating) complex formationbetween the receptor protein and the hedgehog polypeptide. The efficacyof the compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. In the control assay, isolated and purified hedgehogpolypeptide is added to the receptor protein, and the formation ofreceptor/hedgehog complex is quantitated in the absence of the testcompound.

In other embodiments, a ptc therapeutic of the present invention is onewhich disrupts the association of patched with smoothened.

Agonist and antagonists of cell growth can be distinguished, and theefficacy of the compound can be assessed, by subsequent testing withcertain lung cells, e.g., in culture.

In an illustrative embodiment, the polypeptide utilized as a hedgehogreceptor can be generated from the patched protein. Accordingly, anexemplary screening assay includes all or a suitable portion of thepatched protein which can be obtained from, for example, the humanpatched gene (GenBank U43148) or other vertebrate sources (see GenBankAccession numbers U40074 for chicken patched and U46155 for mousepatched), as well as from drosophila (GenBank Accession number M28999)or other invertebrate sources. The patched protein can be provided inthe screening assay as a whole protein (preferably expressed on thesurface of a cell), or alternatively as a fragment of the full lengthprotein which binds to hedgehog polypeptides, e.g., as one or both ofthe substantial extracellular domains (e.g. corresponding to residuesAsn120-Ser438 and/or Arg770-Trp1027 of the human patched protein—whichare also potential antagonists of hedgehog-dependent signaltransduction). For instance, the patched protein can be provided insoluble form, as for example a preparation of one of the extracellulardomains, or a preparation of both of the extracellular domains which arecovalently connected by an unstructured linker (see, for example, Hustonet al. (1988) PNAS 85:4879; and U.S. Pat. No. 5,091,513). In otherembodiments, the protein can be provided as part of a liposomalpreparation or expressed on the surface of a cell. The patched proteincan derived from a recombinant gene, e.g., being ectopically expressedin a heterologous cell. For instance, the protein can be expressed onoocytes, mammalian cells (e.g., COS, CHO, 3T3 or the like), or yeastcell by standard recombinant DNA techniques. These recombinant cells canbe used for receptor binding, signal transduction or gene expressionassays. Mango et al. (1996) Development 122:1225-1233 illustrates abinding assay of human hedgehog to chick patched protein ectopicallyexpressed in Xenopus laevis oocytes. The assay system of Marigo et al.can be adapted to the present drug screening assays. As illustrated inthat reference, Shh binds to the patched protein in a selective,saturable, dose-dependent manner, thus demonstrating that patched is areceptor for Shh.

Complex formation between the hedgehog polypeptide and a hedgehogreceptor may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labelled proteins such as radiolabelled,fluorescently labelled, or enzymatically labelled hedgehog polypeptides,by immunoassay, or by chromatographic detection.

Typically, for cell-free assays, it will be desirable to immobilizeeither the hedgehog receptor or the hedgehog polypeptide to facilitateseparation of receptor/hedgehog complexes from uncomplexed forms of oneof the proteins, as well as to accommodate automation of the assay. Inone embodiment, a fusion protein can be provided which adds a domainthat allows the protein to be bound to a matrix. For example,glutathione-S-transferase/receptor (GST/receptor) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the hedgehog polypeptide, e.g. an ³⁵S-labeled hedgehogpolypeptide, and the test compound and incubated under conditionsconducive to complex formation, e.g. at physiological conditions forsalt and pH, though slightly more stringent conditions may be desired.Following incubation, the beads are washed to remove any unboundhedgehog polypeptide, and the matrix bead-bound radiolabel determineddirectly (e.g. beads placed in scintillant), or in the supernatant afterthe receptor/hedgehog complexes are dissociated. Alternatively, thecomplexes can be dissociated from the bead, separated by SDS-PAGE gel,and the level of hedgehog polypeptide found in the bead fractionquantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, soluble portionsof the hedgehog receptor protein can be immobilized utilizingconjugation of biotin and streptavidin. For instance, biotinylatedreceptor molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with the hedgehog receptorbut which do not interfere with hedgehog binding can be derivatized tothe wells of the plate, and the receptor trapped in the wells byantibody conjugation. As above, preparations of a hedgehog polypeptideand a test compound are incubated in the receptor-presenting wells ofthe plate, and the amount of receptor/hedgehog complex trapped in thewell can be quantitated. Exemplary methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thehedgehog polypeptide, or which are reactive with the receptor proteinand compete for binding with the hedgehog polypeptide; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the hedgehog polypeptide. In the instance of the latter,the enzyme can be chemically conjugated or provided as a fusion proteinwith the hedgehog polypeptide. To illustrate, the hedgehog polypeptidecan be chemically cross-linked or genetically fused with alkalinephosphatase, and the amount of hedgehog polypeptide trapped in thecomplex can be assessed with a chromogenic substrate of the enzyme, e.g.paranitrophenylphosphate. Likewise, a fusion protein comprising thehedgehog polypeptide and glutathione-S-transferase can be provided, andcomplex formation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asthe anti-hedgehog antibodies described herein, can be used.Alternatively, the protein to be detected in the complex can be “epitopetagged” in the form of a fusion protein which includes, in addition tothe hedgehog polypeptide or hedgehog receptor sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, NJ).

Where the desired portion of the hedgehog receptor (or other hedgehogbinding molecule) cannot be provided in soluble form, liposomal vesiclescan be used to provide manipulatable and isolatable sources of thereceptor. For example, both authentic and recombinant forms of thepatched protein can be reconstituted in artificial lipid vesicles (e.g.phosphatidylcholine liposomes) or in cell membrane-derived vesicles(see, for example, Bear et al. (1992) Cell 68:809-818; Newton et al.(1983) Biochemistry 22:6110-6117; and Reber et al. (1987) J Biol Chem262:11369-11374).

In addition to cell-free assays, such as described above, the readilyavailable source of hedgehog proteins provided by the art alsofacilitates the generation of cell-based assays for identifying smallmolecule agonists/antagonists and the like. Analogous to the cell-basedassays described above for screening combinatorial libraries, cellswhich are sensitive to hedgehog induction, e.g. patched-expressing cellsor other lung-derived cells sensitive to hedgehog induction, can becontacted with a hedgehog protein and a test agent of interest, with theassay scoring for anything from simple binding to the cell to modulationin hedgehog inductive responses by the target cell in the presence andabsence of the test agent. As with the cell-free assays, agents whichproduce a statistically significant change in hedgehog activities(either inhibition or potentiation) can be identified.

In other embodiments, the cell-based assay scores for agents whichdisrupt association of patched and smoothened proteins, e.g., in thecell surface membrane or liposomal preparation.

In addition to characterizing cells that naturally express the patchedprotein, cells which have been genetically engineered to ectopicallyexpress patched can be utilized for drug screening assays. As anexample, cells which either express low levels or lack expression of thepatched protein, e.g. Xenopus laevis oocytes, COS cells or yeast cells,can be genetically modified using standard techniques to ectopicallyexpress the patched protein. (see Marigo et al., supra).

The resulting recombinant cells, e.g., which express a functionalpatched receptor, can be utilized in receptor binding assays to identifyagonist or antagonists of hedgehog binding. Binding assays can beperformed using whole cells. Furthermore, the recombinant cells of thepresent invention can be engineered to include other heterologous genesencoding proteins involved in hedgehog-dependent signal pathways. Forexample, the gene products of one or more of smoothened, costal-2 and/orfused can be co-expressed with patched in the reagent cell, with assaysbeing sensitive to the functional reconstitution of the hedgehog signaltransduction cascade.

Alternatively, liposomal preparations using reconstituted patchedprotein can be utilized. Patched protein purified from detergentextracts from both authentic and recombinant origins can bereconstituted in in artificial lipid vesicles (e.g. phosphatidylcholineliposomes) or in cell membrane-derived vesicles (see, for example, Bearet al. (1992) Cell 68:809-818; Newton et al. (1983) Biochemistry22:6110-6117; and Reber et al. (1987) J Biol Chem 262:11369-11374). Thelamellar structure and size of the resulting liposomes can becharacterized using electron microscopy. External orientation of thepatched protein in the reconstituted membranes can be demonstrated, forexample, by immunoelectron microscopy. The hedgehog protein bindingactivity of liposomes containing patched and liposomes without theprotein in the presence of candidate agents can be compared in order toidentify potential modulators of the hedgehog-patched interaction.

The hedgehog protein used in these cell-based assays can be provided asa purified source (natural or recombinant in origin), or in the form ofcells/tissue which express the protein and which are co-cultured withthe target cells. As in the cell-free assays, where simple binding(rather than induction) is the hedgehog activity scored for in theassay, the protein can be labelled by any of the above-mentionedtechniques, e.g., fluorescently, enzymatically or radioactively, ordetected by immunoassay.

In addition to binding studies, functional assays can be used toidentified modulators, i.e., agonists or antagonists, of hedgehog orpatched activities. By detecting changes in intracellular signals, suchas alterations in second Messengers or gene expression, inpatched-expressing cells contacted with a test agent, candidate agonistsand antagonists to patched signaling can be identified.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, the transcription factor cubitusinterruptus (ci), the serine/threonine kinase fused (fu) and the geneproducts of costal-2, smoothened and suppressor of fused.

The interaction of a hedgehog protein with patched sets in motion acascade involving the activation and inhibition of downstream effectors,the ultimate consequence of which is, in some instances, a detectablechange in the transcription or translation of a gene. Potentialtranscriptional targets of patched signaling are the patched gene itself(Hidalgo and Ingham, 1990 Development 110, 291-301; Mango et al., 1996)and the vertebrate homologs of the drosophila cubitus interruptus gene,the GLI genes (Hui et al. (1994) Dev Biol 162:402-413). Patched geneexpression has been shown to be induced in cells of the limb bud and theneural plate that are responsive to Shh. (Mango et al. (1996) PNAS;Mango et al. (1996) Development 122:1225-1233). The GLI genes encodeputative transcription factors having zinc finger DNA binding domains(Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) MolCell Biol 10:634-642). Transcription of the GLI gene has been reportedto be upregulated in response to hedgehog in limb buds, whiletranscription of the GLI3 gene is downregulated in response to hedgehoginduction (Mango et al. (1996) Development 122:1225-1233). By selectingtranscriptional regulatory sequences from such target genes, e.g. frompatched or GLI genes, that are responsible for the up- or downregulation of these genes in response to patched signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify patched signalling pathways. Expressionof the reporter gene, thus, provides a valuable screening tool for thedevelopment of compounds that act as agonists or antagonists of ptcinduction of differentiation/quiescence.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc signaling. To identify potentialregulatory elements responsive to ptc signaling present in thetranscriptional regulatory sequence of a target gene, nested deletionsof genomic clones of the target gene can be constructed using standardtechniques. See, for example, Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989); U.S.Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem 270:10314-10322; andKube et al. (1995) Cytokine 7:1-7. A nested set of DNA fragments fromthe gene's 5′-flanking region are placed upstream of a reporter gene,such as the luciferase gene, and assayed for their ability to directreporter gene expression in patched expressing cells. Host cellstransiently transfected with reporter gene constructs can be scored forthe induction of expression of the reporter gene in the presence andabsence of hedgehog to determine regulatory sequences which areresponsice to patched-dependent signalling.

In practicing one embodiment of the assay, a reporter gene construct isinserted into the reagent cell in order to generate a detection signaldependent on second messengers generated by induction with hedgehogprotein. Typically, the reporter gene construct will include a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the hedgehog activity, with the level ofexpression of the reporter gene providing the hedgehog-dependentdetection signal. The amount of transcription from the reporter gene maybe measured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic activity. The amount of expression from the reporter gene isthen compared to the amount of expression in either the same cell in theabsence of the test compound (or hedgehog) or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testcompound has in some manner altered the signal transduction of thepatched protein, e.g., the test compound is a potential ptc therapeutic.

As described in further detail below, in preferred embodiments the geneproduct of the reporter is detected by an intrinsic activity associatedwith that product. For instance, the reporter gene may encode a geneproduct that, by enzymatic activity, gives rise to a detection signalbased on color, fluorescence, or luminescence. In other preferredembodiments, the reporter or marker gene provides a selective growthadvantage, e.g., the reporter gene may enhance cell viability, relieve acell nutritional requirement, and/or provide resistance to a drug.

Preferred reporter genes are those that are readily detectable. Thereporter gene may also be included in the construct in the form of afusion gene with a gene that includes desired transcriptional regulatorysequences or exhibits other desirable properties. Examples of reportergenes include, but are not limited to CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase,and other enzyme detection systems, such as beta-galactosidase; fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterialluciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwinet al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992) Methods in Enzymol. 216:362-368).

Transcriptional control elements which may be included in a reportergene construct include, but are not limited to, promoters, enhancers,and repressor and activator binding sites. Suitable transcriptionalregulatory elements may be derived from the transcriptional regulatoryregions of genes whose expression is induced after modulation of apatched signal transduction pathway. The characteristics of preferredgenes from which the transcriptional control elements are derivedinclude, but are not limited to, low or undetectable expression inquiescent cells, rapid induction at the transcriptional level withinminutes of extracellular simulation, induction that is transient andindependent of new protein synthesis, subsequent shut-off oftranscription requires new protein synthesis, and mRNAs transcribed fromthese genes have a short half-life. It is not necessary for all of theseproperties to be present.

In yet other embodiments, second messenger generation can be measureddirectly in the detection step, such as mobilization of intracellularcalcium, phospholipid metabolism or adenylate cyclase activity arequantitated, for instance, the products of phospholipid hydrolysis IP₃,DAG or cAMP could be measured For example, recent studies haveimplicated protein kinase A (PKA) as a possible component ofhedgehog/patched signaling (Hammerschmidt et al. (1996) Genes & Dev10:647). High PKA activity has been shown to antagonize hedgehogsignaling in these systems. Although it is unclear whether PKA actsdirectly downstream or in parallel with hedgehog signaling, it ispossible that hedgehog signalling occurs via inhibition of PKA activity.Thus, detection of PKA activity provides a potential readout for theinstant assays.

In a preferred embodiment, the ptc therapeutic is a PKA inhibitor. Avariety of PKA inhibitors are known in the art, including both peptidyland organic compounds. For instance, the ptc therapeutic can be a5-isoquinolinesulfonamide, such as represented in the general formula:

wherein,

R₁ and R₂ each can independently represent hydrogen, and as valence andstability permit a lower alkyl, a lower alkenyl, a lower alkynyl, acarbonyl (such as a carboxyl, an ester, a formate, or a ketone), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₈, or

R₁ and R₂ taken together with N form a heterocycle (substituted orunsubstituted);

R₃ is absent or represents one or more substitutions to the isoquinolinering such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₈;

R₈ represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and

n and m are independently for each occurrence zero or an integer in therange of 1 to 6.

In a preferred embodiment, the PKA inhibitor isN-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89;Calbiochem Cat. No. 371963), e.g., having the formula:

In another embodiment, the PKA inhibitor is1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7; Calbiochem Cat. No.371955), e.g., having the formula:

In still other embodiments, the PKA inhibitor is KT5720 (Calbiochem Cat.No. 420315), having the structure

A variety of nucleoside analogs are also useful as PKA inhibitors. Forexample, the subject method can be carried out cyclic AMP analogs whichinhibit the kinase activity of PKA, as for example, 8-bromo-cAMP ordibutyryl-cAMP

Exemplary peptidyl inhibitors of PICA activity include the PICA HeatStable Inhibitor (isoform α; see, for example, Calbiochem Cat. No.539488, and Wen et al. (1995) J Biol Chem 270:2041).

Certain hedgehog receptors may stimulate the activity of phospholipases.Inositol lipids can be extracted and analyzed using standard lipidextraction techniques. Water soluble derivatives of all three inositollipids (IP₁, IP₂; IP₃) can also be quantitated using radiolabellingtechniques or HPLC.

The mobilization of intracellular calcium or the influx of calcium fromoutside the cell may be a response to hedgehog stimulation or lack thereof. Calcium flux in the reagent cell can be measured using standardtechniques. The choice of the appropriate calcium indicator,fluorescent, bioluminescent, metallochromic, or Ca⁺⁺-sensitivemicroelectrodes depends on the cell type and the magnitude and timeconstant of the event under study (Borle (1990) Environ Health Perspect84:45-56). As an exemplary method of Ca⁺⁺ detection, cells could beloaded with the Ca⁺⁺ sensitive fluorescent dye fura-2 or indo-1, usingstandard methods, and any change in Ca⁺⁺ measured using a fluorometer.

In certain embodiments of the assay, it may be desirable to screen forchanges in cellular phosphorylation. As an example, the drosophila genefused (fu) which encodes a serine/threonine kinase has been identifiedas a potential downstream target in hedgehog signaling. (Preat et al.,1990 Nature 347, 87-89; Therond et al. 1993, Mech. Dev. 44. 65-80). Theability of compounds to modulate serine/threonine kinase activationcould be screened using colony immunoblotting (Lyons and Nelson (1984)Proc. Natl. Acad. Sci. USA 81:7426-7430) using antibodies againstphosphorylated serine or threonine residues. Reagents for performingsuch assays are commercially available, for example, phosphoserine andphosphothreonine specific antibodies which measure increases inphosphorylation of those residues can be purchased from commercialsources.

In yet another embodiment, the ptc therapeutic is an antisense moleculewhich inhibits expression of a protein involved in a patched-mediatedsignal transduction pathway. To illustrate, by inhibiting the expressionof a protein which are involved in patched signals, such as fused,costal-2, smoothened and/or Gli genes, the ability of the patched signalpathway(s) to inhibit proliferation of a cell can be altered, e.g.,potentiated or repressed.

As used herein, “antisense” therapy refers to administration or in situgeneration of oligonucleotide probes or their derivatives whichspecifically hybridize (e.g. bind) under cellular conditions withcellular mRNA and/or genomic DNA encoding a hedgehog protein, patched,or a protein involved in patched-mediated signal transduction. Thehybridization should inhibit expression of that protein, e.g. byinhibiting transcription and/or translation. The binding may be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interactions in the majorgroove of the double helix. In general, “antisense” therapy refers tothe range of techniques generally employed in the art, and includes anytherapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thetarget cellular mRNA. Alternatively, the antisense construct is anoligonucleotide probe which is generated ex vivo and which, whenintroduced into the cell causes inhibition of expression by hybridizingwith the mRNA and/or genomic sequences of a target gene. Sucholigonucleotide probes are preferably modified oligonucleotide which areresistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and is therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al. (1988) Biotechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

Several considerations should be taken into account when constructingantisense oligonucleotides for the use in the methods of the invention:(1) oligos should have a GC content of 50% or more; (2) avoid sequenceswith stretches of 3 or more G's; and (3) oligonucleotides should not belonger than 25-26 mers. When testing an antisense oligonucleotide, amismatched control can be constructed. The controls can be generated byreversing the sequence order of the corresponding antisenseoligonucleotide in order to conserve the same ratio of bases.

In an illustrative embodiment, the ptc therapeutic can be an antisenseconstruct for inhibiting the expression of patched, e.g., to mimic theinhibition of patched by hedgehog. Exemplary antisense constructsinclude:

5′-GTCCTGGCGCCGCCGCCGCCGTCGCC 5′-TTCCGATGACCGGCCTTTCGCGGTGA5′-GTGCACGGAAAGGTGCAGGCCACACT

VI. Exemplary Pharmaceutical Preparations of Hedgehog and PtcTherapeutics

The source of the hedgehog and ptc therapeutics to be formulated willdepend on the particular form of the agent. Small organic molecules andpeptidyl fragments can be chemically synthesized and provided in a pureform suitable for pharmaceutical/cosmetic usage. Products of naturalextracts can be purified according to techniques known in the art. Forexample, the Cox et al. U.S. Pat. No. 5,286,654 describes a method forpurifying naturally occurring forms of a secreted protein and can beadapted for purification of hedgehog polypeptides. Recombinant sourcesof hedgehog polypeptides are also available. For example, the geneencoding hedgehog polypeptides, are known, inter alia, from PCTpublications WO 95/18856 and WO 96/17924.

Those of skill in treating lung tissues can determine the effectiveamount of an ptc, hedgehog or fgf-10 therapeutic to be formulated in apharmaceutical or cosmetic preparation.

The ptc, hedgehog or fgf-10 therapeutic formulations used in the methodof the invention are most preferably applied in the form of appropriatecompositions. As appropriate compositions there may be cited allcompositions usually employed for systemically or topicallyadministering drugs. The pharmaceutically acceptable carrier should besubstantially inert, so as not to act with the active component.Suitable inert carriers include water, alcohol polyethylene glycol,mineral oil or petroleum gel, propylene glycol and the like.

To prepare the pharmaceutical compositions of this invention, aneffective amount of the particular ptc, hedgehog or fgf-10 therapeuticas the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirable inunitary dosage form suitable, particularly, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules, and tablets. Because of their ease in administration, tabletsand capsules represents the most advantageous oral dosage unit form, inwhich case solid pharmaceutical carriers are obviously employed. Forparenteral compositions, the carrier will usually comprise sterilewater, at least in large part, though other ingredients, for example, toaid solubility, may be included. Injectable solutions, for example, maybe prepared in which the carrier comprises saline solution, glucosesolution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed. Also includedare solid form preparations which are intended to be converted, shortlybefore use, to liquid form preparations. In the compositions suitablefor percutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not introduce a significant deleterious effect on theskin.

In addition to the direct topical application of the preparations theycan be topically administered by other methods, for example,encapsulated in a temperature and/or pressure sensitive matrix or infilm or solid carrier which is soluble in body fluids and the like forsubsequent release, preferably sustained-release of the activecomponent.

As appropriate compositions for topical application there may be citedall compositions usually employed for topically administeringtherapeutics, e.g., creams, gellies, dressings, shampoos, tinctures,pastes, ointments, salves, powders, liquid or semiliquid formulation andthe like. Application of said compositions may be by aerosol e.g. with apropellent such as nitrogen carbon dioxide, a freon, or without apropellent such as a pump spray, drops, lotions, or a semisolid such asa thickened composition which can be applied by a swab. In particularcompositions, semisolid compositions such as salves, creams, pastes,gellies, ointments and the like will conveniently be used.

It is especially advantageous to formulate the subject compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used in the specification and claims herein refersto physically discreate units suitable as unitary dosages, each unitcontaining a predetermined quantity of active ingredient calculated toproduce the to desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such dosage unit forms aretablets (including scored or coated tablets), capsules, pills, powderspackets, wafers, injectable solutions or suspensions, teaspoonfuls,tablespoonfuls and the like, and segregated multiples thereof.

The pharmaceutical preparations of the present invention can be used, asstated above, for the many applications which can be considered cosmeticuses. Cosmetic compositions known in the art, preferably hypoallergenicand pH controlled are especially preferred, and include toilet waters,packs, lotions, skin milks or milky lotions. The preparations contain,besides the ptc, hedgehog or fgf-10 therapeutic, components usuallyemployed in such preparations. Examples of such components are oils,fats, waxes, surfactants, humectants, thickening agents, antioxidants,viscosity stabilizers, chelating agents, buffers, preservatives,perfumes, dyestuffs, lower alkanols, and the like. If desired, furtheringredients may be incorporated in the compositions, e.g.antiinflammatory agents, antibacterials, antifungals, disinfectants,vitamins, sunscreens, antibiotics, or other anti-acne agents.

Examples of oils comprise fats and oils such as olive oil andhydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons suchas liquid paraffin, ceresin, and squalane; fatty acids such as stearicacid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol,lanolin alcohol, and hexadecanol; and esters such as isopropylmyristate, isopropyl palmitate and butyl stearate. As examples ofsurfactants there may be cited anionic surfactants such as sodiumstearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate,sodium N-acyl glutamate; cationic surfactants such asstearyldimethylbenzylammonium chloride and stearyltrimethylammoniumchloride; ampholytic surfactants such as alkylaminoethylglycinehydrocloride solutions and lecithin; and nonionic surfactants such asglycerin monostearate, sorbitan monostearate, sucrose fatty acid esters,propylene glycol monostearate, polyoxyethylene oleylether, polyethyleneglycol monostearate, polyoxyethylene sorbitan monopalmitate,polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropyleneglycol (e.g. the materials sold under the trademark “Pluronic”),polyoxyethylene castor oil, and polyoxyethylene lanolin. Examples ofhumectants include glycerin, 1,3-butylene glycol, and propylene glycol;examples of lower alcohols include ethanol and isopropanol; examples ofthickening agents include xanthan gum, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, polyethylene glycol and sodiumcarboxymethyl cellulose; examples of antioxidants comprise butylatedhydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acidand ethoxyquin; examples of chelating agents include disodium edetateand ethanehydroxy diphosphate; examples of buffers comprise citric acid,sodium citrate, boric acid, borax, and disodium hydrogen phosphate; andexamples of preservatives are methyl parahydroxybenzoate, ethylparahydroxybenzoate, dehydroacetic acid, salicylic acid and benzoicacid.

For preparing ointments, creams, toilet waters, skin milks, and thelike, typically from 0.01 to 10% in particular from 0.1 to 5% and morein particular from 0.2 to 2.5% of the active ingredient, e.g., of theptc, hedgehog or fgf-10 therapeutic, will be incorporated in thecompositions. In ointments or creams, the carrier for example consistsof 1 to 20%, in particular 5 to 15% of a humectant, 0.1 to 10% inparticular from 0.5 to 5% of a thickener and water; or said carrier mayconsist of 70 to 99%, in particular 20 to 95% of a surfactant, and 0 to20%, in particular 2.5 to 15% of a fat; or 80 to 99.9% in particular 90to 99% of a thickener; or 5 to 15% of a surfactant, 2-15% of ahumectant, 0 to 80% of an oil, very small (<2%) amounts of preservative,coloring agent and/or perfume, and water. In a toilet water, the carrierfor example consists of 2 to 10% of a lower alcohol, 0.1 to 10% or inparticular 0.5 to 1% of a surfactant, 1 to 20%, in particular 3 to 7% ofa humectant, 0 to 5% of a buffer, water and small amounts (<2%) ofpreservative, dyestuff and/or perfume. In a skin milk, the carriertypically consists of 10-50% of oil, 1 to 10% of surfactant, 50-80% ofwater and 0 to 3% of preservative and/or perfume. In the aforementionedpreparations, all % symbols refer to weight by weight percentage.

Particular compositions for use in the method of the present inventionare those wherein the ptc, hedgehog or fgf-10 therapeutic is formulatedin liposome-containing compositions. Liposomes are artificial vesiclesformed by amphiphatic molecules such as polar lipids, for example,phosphatidyl cholines, ethanolamines and serines, sphingomyelins,cardiolipins, plasmalogens, phosphatidic acids and cerebiosides.Liposomes are formed when suitable amphiphathic molecules are allowed toswell in water or aqueous solutions to form liquid crystals usually ofmultilayer structure comprised of many bilayers separated from eachother by aqueous material (also referred to as coarse liposomes).Another type of liposome known to be consisting of a single bilayerencapsulating aqueous material is referred to as a unilamellar vesicle.If water-soluble materials are included in the aqueous phase during theswelling of the lipids they become entrapped in the aqueous layerbetween the lipid bilayers.

Water-soluble active ingredients such as, for example, various saltforms of a hedgehog polypeptide, are encapsulated in the aqueous spacesbetween the molecular layers. The lipid soluble active ingredient ofptc, hedgehog or fgf-10 therapeutic, such as an organic mimetic, ispredominantly incorporated into the lipid layers, although polar headgroups may protude from the layer into the aqueous space. Theencapsulation of these compounds can be achieved by a number of methods.The method most commonly used involves casting a thin film ofphospholipid onto the walls of a flask by evaporation from an organicsolvent. When this film is dispersed in a suitable aqueous medium,multilamellar liposomes are formed. Upon suitable sonication, the coarseliposomes form smaller similarly closed vesicles.

Water-soluble active ingredients are usually incorporated by dispersingthe cast film with an aqueous solution of the compound. Theunencapsulated compound is then removed by centrifugation,chromatography, dialysis or other art-known suitable procedures. Thelipid-soluble active ingredient is usually incorporated by dissolving itin the organic solvent with the phospholipid prior to casting the film.If the solubility of the material in the lipid phase is not exceeded orthe amount present is not in excess of that which can be bound to thelipid, liposomes prepared by the above method usually contain most ofthe material bound in the lipid bilayers; separation of the liposomesfrom unencapsulated material is not required.

A particularly convenient method for preparing liposome formulated formsof hedgehog and ptc therapeutics is the method described inEP-A-253,619, incorporated herein by reference. In this method, singlebilayered liposomes containing encapsulated active ingredients areprepared by dissolving the lipid component in an organic medium,injecting the organic solution of the lipid component under pressureinto an aqueous component while simultaneously mixing the organic andaqueous components with a high speed homogenizer or mixing means,whereupon the liposomes are formed spontaneously.

The single bilayered liposomes containing the encapsulated ptc, hedgehogor fgf-10 therapeutic can be employed directly or they can be employedin a suitable pharmaceutically acceptable carrier for topicaladministration. The viscosity of the liposomes can be increased by theaddition of one or more suitable thickening agents such as, for examplexanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose andmixtures thereof. The aqueous component may consist of water alone or itmay contain electrolytes, buffered systems and other ingredients, suchas, for example, preservatives. Suitable electrolytes which can beemployed include metal salts such as alkali metal and alkaline earthmetal salts. The preferred metal salts are calcium chloride, sodiumchloride and potassium chloride. The concentration of the electrolytemay vary from zero to 260 mM, preferably from 5 mM to 160 mM. Theaqueous component is placed in a suitable vessel which can be adapted toeffect homogenization by effecting great turbulence during the injectionof the organic component. Homogenization of the two components can beaccomplished within the vessel, or, alternatively, the aqueous andorganic components may be injected separately into a mixing means whichis located outside the vessel. In the latter case, the liposomes areformed in the mixing means and then transferred to another vessel forcollection purpose.

The organic component consists of a suitable non-toxic, pharmaceuticallyacceptable solvent such as, for example ethanol, glycerol, propyleneglycol and polyethylene glycol, and a suitable phospholipid which issoluble in the solvent. Suitable phospholipids which can be employedinclude lecithin, phosphatidylcholine, phosphatydylserine,phosphatidylethanol-amine, phosphatidylinositol, lysophosphatidylcholineand phospha-tidyl glycerol, for example. Other lipophilic additives maybe employed in order to selectively modify the characteristics of theliposomes. Examples of such other additives include stearylamine,phosphatidic acid, tocopherol, cholesterol and lanolin extracts.

In addition, other ingredients which can prevent oxidation of thephospholipids may be added to the organic component. Examples of suchother ingredients include tocopherol, butylated hydroxyanisole,butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.Preservatives such a benzoic acid, methyl paraben and propyl paraben mayalso be added.

Apart from the above-described compositions, use may be made of covers,e.g. plasters, bandages, dressings, gauze pads and the like, containingan appropriate amount of a ptc, hedgehog or fgf-10 therapeutic. In somecases use may be made of plasters, bandages, dressings, gauze pads andthe like which have been impregnated with a topical formulationcontaining the therapeutic formulation.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

The mammalian lung, like many other organs, develops by branchingmorphogenesis of an epithelium [see ref. 1]. Development initiates withevagination of two ventral buds of foregut endoderm into the underlyingsplanchnic mesoderm. As they extend, they send out lateral branches atprecise, invariant positions establishing the primary airways and thelobes of each lung. Dichotomous branching leads to further extension ofthe airways. Grafting studies have demonstrated the importance ofbronchial mesenchyme in inducing epithelial branching, but thesignificance of epithelial signaling is largely unstudied. The morphogenSonic hedgehog (Shh) is widely expressed in the foregut endoderm and isspecifically up-regulated in the distal epithelium of the lung wherebranching is occurring [see ref. 2]. Ectopic expression of Shh disruptsbranching and increases proliferation suggesting that local Shhsignaling regulates lung development [see ref. 2]. We report here thatShh is essential for development of the respiratory system. In Shh nullmutants, the trachea and esophagus do not separate properly and thelungs form a rudimentary sac due to failure of branching and growthafter formation of the primary lung buds. Interestingly, normalproximo-distal differentiation of the airway epithelium occurs,indicating that Shh is not needed for differentiation events. Inaddition, the transcription of several mesenchymally expresseddownstream targets of Shh is abolished. These results highlight theimportance of epithelially derived Shh in regulating branchingmorphogenesis of the lung.

Results and Discussion

To address the role of Shh in respiratory tract development, we examineda null mutant of the gene (3). At 10.5 days post coitum (dpc) ofembryonic mouse development, the lung of wild-type (wt) siblingsconsists of a left and right bud [see ref 1]. By 12.5 dpc, the tracheaepithelium has separated ventrally from the esophageal component of theforegut and the two lung buds have formed several lateral branches whichwill give rise to primary airways of the lung lobes (FIG. 1 a-c). Incontrast, the esophageal and tracheal tubes remain closely associated inShh mutants (FIG. 1 d,e) and although left and right buds form, theyeither have not branched or possess one abnormally positioned branchpoint (FIG. 10. Wild-type lungs undergo considerable growth andbranching in organ culture. However, in explant culture of lungs fromShh mutants, bronchial mesenchyme cells detach from the endoderm and theepithelium fails to grow, or branch extensively (data not shown). Weconclude that the defect in branching morphogenesis is independent ofother Shh-expressing organs (i.e., the gut), and that the observedbranching phenotype reflects an absence of Shh signaling which isnormally associated with the branching process.

To determine if branching is merely delayed and whether Shh plays a rolein differentiation, we examined lungs removed at 15.5 (data not shown)and 18.5 dpc (FIG. 1 g,h). At this time, five well-developed lobes areevident in the wild-type (four right, one left), and highly branchedairways form a ramifying epithelial network, the respiratory tree (FIG.1 i,k,l). To mediate gas exchange in the alveolar sacs, the respiratorysurface is well vascularized (FIG. 1 g). In contrast, Shh mutants formonly a rudimentary respiratory organ with a few large, poorlyvascularized airways (FIG. 1 h). Trachea and esophagus are so closelyjuxtaposed that their tubes share some common epithelium (FIG. 1 e) anda fistula-like fusion of the alimentary and respiratory tract is formed,mirroring a lethal anomaly well described in human pathology [see ref.4,5] (FIG. 1 j,m).

Remarkably, despite the absence of branching, evidence of normalproximo-distal epithelial differentiation can be observed. Mostproximally, the pulmonary epithelium forms a columnar epithelium typicalof the mainstem bronchi (FIG. 1 m) and expresses CCSP [see ref. 6], amarker for terminally differentiated secretory Clara cells (FIG. 1 q).More distally, the epithelium consists of a mixture of columnar andcuboidal epithelium as observed in the bronchioles (FIG. 1 n), andalveolar air sacs are formed which correspondingly express SP—C [seeref. 7], a type II pneumocyte marker (FIG. 1 r).

In summary, Shh is not required for proximo-distal differentiation oflung epithelium, but is essential for three different events of regionalmorphogenesis of the foregut endoderm, formation of thetracheoesophageal septum, lung lobation and generation of therespiratory tree, all of which are essential in forming a functionallung.

The exact role for Shh in branching processes remains to be determined.Grafting studies indicate that, whereas budding can be supported bymesenchyme from many different sources, only bronchial mesenchyme caninduce organotypic branching morphogenesis [see ref. 8]. The requirementfor Shh in the epithelium suggests that regulation of its expression maybe a reciprocal epithelial response to mesenchymal signaling.

To examine in more detail how Shh might regulate early branching of thelung epithelium, we performed digoxigenin in situ hybridization withprobes recognizing general targets of Hedgehog signaling (FIG. 2 a-e anddata not shown), or genes specifically implicated in lung morphogenesis(FIG. 2 f-k). As Shh mutants are growth retarded and show a generaldelay in lung budding, we compared expression of these markers at 12.5dpc with wild type embryos collected at 11.5 and 12.5 dpc.

Patched genes encode proteins thought to be Hedgehog receptors, whileGli-genes encode transcriptional mediators of Hedgehog signaling [seeref. 9]. Both Ptc-1 and Gli-1 are up-regulated when Shh is ectopicallyexpressed in the lung indicating that here, as elsewhere in the embryo,they are transcriptional targets of Shh signaling [see ref. 9,10].Consistent with this model, Ptc-1 and Gli-1 are normally expressed inthe mesenchyme of wild-type embryos with highest levels at the distalbranch points mirroring epithelial Shh expression [see ref. 10] (FIG. 2a,c). In Shh mutants, only basal levels of expression of both genes aredetected (FIG. 2 a,c). Gli-3 which shows more wide-spread expression inthe mesenchyme is also down-regulated (FIG. 2 e). In contrast, Ptc-2which is expressed at higher levels in the epithelium and Gli-2, whichis normally expressed more uniformly in the mesenchyme are not altered(FIG. 2 b,d). These data indicate that the lung mesenchyme, not theepithelium, is most likely the direct cellular target of Shh signaling.Further, they suggest that modulation of Gli-1 and Gli-3 transcriptionmay be a critical aspect of lung morphogenesis. As Gli-1 mutants do nothave a lung phenotype, the Shh phenotype cannot simply be ascribed to aloss of Gli-1 transcriptional activity [see ref. 1.0]. Given thatpost-transcriptional processing regulates Gli (Ci) activity ininvertebrates [see ref. 11], we cannot rule out that Gli-2 is expressed,but posttranscriptionally inactivated. Gli genes are clearly involved inlung development, as evidenced by the relatively weak lobular hypoplasiaobserved in Gli-3 mutants [see ref. 10], but revealing the full extentof Gli action may require the generation of compound mutants.

Several lines of evidence indicate that hedgehog signaling regulates theexpression of Bmp, Wnt and FGF family members [see ref. 11]. In thelung, Bmp-4 is strongly expressed in the distal-most tips of theepithelium. Ectopic expression results in decreased epithelialproliferation, disrupted branching and reduced differentiation of distalcell types in the airway [see ref. 12]. In Shh mutants, Bmp 4 isexpressed in the normal position but at higher levels (FIG. 2 f),suggesting that enhanced Bmp 4 signaling could contribute to the blockin branching. Wnt-7b is normally expressed in the lung epithelium and isrequired for normal branching (S. Lee, W. Cardoso, B. Parr & A. McMahon;unpublished), whereas Wnt-2 is expressed in the underlying mesenchymesuggesting a role in epithelial maintenance [see ref 2]. In Shh mutants,Wnt-7b expression is not altered (FIG. 2 g) but Wnt-2 expression isdown-regulated (FIG. 2 h). This observation lends further support to themodel that the lung mesenchyme is the primary target of Shh signalingand indicates that mesenchymal signaling is abnormal in Shh mutants.However, no role for Wnt-2 in lung development has been reported inWnt-2 mutants [see ref 13].

Ectopic expression of a dominant negative form of FGF-R2 in the lungepithelium arrests branching after formation of left and right budswhich then grow caudally as tubes, differentiating into proximalepithelial structures only [see ref. 14]. An arrest in branching afterinitial budding is reminiscent of Shh mutants, but there are clearlydifferences in subsequent morphogenesis and differentiation which islargely unaffected in Shh mutants. The recent observation that Fgf70 isexpressed in mesenchyme cells preceding branch formation and can inducebranching of lung epithelium in culture, points to its role as aputative ligand [see ref. 15]. In Shh mutants, expression of FGF-R2 isunaltered (FIG. 21). In contrast, Fgf70 which in wild-type embryos ishighly localized to small patches of mesenchyme at a distance from thelung epithelium (arrows in FIG. 2 j), is expressed broadly in mesenchymeimmediately adjacent to the epithelium in the mutant lung. These resultsindicate that Shh is not required for Fgf70 expression. Further, theysuggest that Shh signaling may spatially restrict Fgf10 expression tothe distal mesenchyme. Such an inhibitory role for Shh in the localregulation of Fgf10 expression is supported by transgenic studies [seeref. 16]. The intriguing possibility that the altered position of Fgf70expression then disrupts branching remains to be determined.

HNF-3β and Nkx-2.1 are specific transcriptional effectors of Shhsignaling in the neural tube. In the gut, HNF-3b is widely expressed inthe epithelium, including the lung, whereas Nkx-2.1 expression isspecific to the lung epithelium and a few other endodermal derivatives[see ref. 17]. Mice lacking Nkx 2.1 develop cystic unbranched lungsindicating that it is essential for lung morphogenesis [see ref. 17].Expression of both genes is unaltered in the epithelium of Shh mutantlungs suggesting that in this organ their expression is independent ofthe Shh signaling pathway (FIG. 2 k and data not shown).

As loss of Shh activity predominantly affects the expression ofmesenchyme markers, we analyzed late mesenchyme differentiation.Formation of cartilage rings, albeit disorganized, occurs in the mutant(FIG. 3 a), while the layer of smooth muscle typically lining theproximal epithelium is absent (FIG. 3 b). The observation that Shh isrequired for formation of smooth muscle is in agreement with previousstudies [see ref. 18].

In summary, the results reported here establish Shh as a regulator offoregut development and more specifically as a key factor in the controlof branching morphogenesis in the mouse lung. They also indicate thatthe genetic control of growth and branching in the lung epithelium ismost likely a complex process involving both epithelial and mesenchymalinteractions at the branch points, and that the downstream targets ofShh signaling in this organ are primarily mesenchymally expressed genes.

Materials and Methods Shh Mutants

Generation of the Shh mutants has been described elsewhere [see ref. 3].Mice homozygous for the null allele appear phenotypically identical tothose reported in [see ref. 19].

Histological/In Situ Analysis

Tissue was processed for standard histology, or a modified in situhybridization procedure [see ref. 20].

Antibody Staining

Antibody staining with a monoclonal antibody against smooth muscle actin(Sigma) was carried out according to the manufacturer's instructions.

REFERENCES CITED IN EXAMPLES

-   1. Ten Have-Opbroek A A W: Lung development in the mouse embryo. Exp    Lung Res 1992, 17:111-130.-   2. Bellusci S et al.: Involvement of Sonic hedgehog (Shh) in mouse    embryonic lung growth and morphogenesis. Development 1997, 124:    53-63.-   3. St.-Jacques B, Dassule H, Karavanova I, Botchkarev V A, Li J,    Danielian P, McMahon J A, Paus R, Lewis P, McMahon A P: Shh    signaling is essential for hair development. Curr Biol., in press.-   4. Sutliff K S, Hutchins G M: Septation of the respiratory and    digestive tracts in human embryos: crucial role of the    tracheoesophageal sulcus. Anatom Record 1994, 238:237-247.-   5. Skandalakis J E et al: The trachea and the lungs. Embr for    Surgeons. 1994, 414-450.-   6. Hackett B P, Gitlin J D: Cell-specific expression of a Clara cell    secretory protein-human growth hormone gene in the bronchiolar    epithelium of transgenic mice. Proc Natl Acad USA 1992,    89:9079-9083.-   7. Bachurski C J, Pryhuber G S, Glasser S W, Kelly S E, Whitsett J    A: Tumor necrosis factor-alpha inhibits surfactant protein C gene    transcription. J Biol Chem 1995, 270:19402-19407.-   8. Spooner B S, Wessells N K Mammalian lung development:    interactions in primordium formation and bronchial morphogenesis. J    Exp Zool 1970, 175: 445-454.-   9. Tabin C J, McMahon A P: Recent advances in hedgehog signaling.    Trends Cell Biol 1997, 7:442-445.-   10. Grindley J C, Bellusci S, Perkins D, Hogan B L M: Evidence for    the involvement of the Gli gene family in embryonic mouse lung    development. Dev Biol 1997, 188: 337-348.-   11. Hammerschmidt M, Brook A, McMahon A P: The world according to    hedgehog. TIGs 1997, 13: 14-21.-   12. Bellusci S, Henderson R, Winnier G, Oikawa T, Hogan B L M:    Evidence from normal expression and targeted misexpression that Bone    Morphogenetic Protein-4 (Bmp-4) plays a role in mouse embryonic lung    morphogenesis. Development 1996, 122: 1693-1702.-   13. Monkley S J, et al.: Targeted disruption of the Wnt2 gene    results in placentation defects. Development 1996, 122: 3343-3353.-   14. Peters K, Werner S, Liao X, Whisett J, Williams S: Targeted    expression of a dominant negative FGF receptor blocks branching    morphogenesis and epithelial differentiation of the mouse lung.    EMBO J. 1996, 13:3296-3301.-   15. Bellusci S. et al.: Fibroblast Growth Factor 10 and branching    morphogenesis in the embryonic mouse lung. Development 1997, 124:    4867-4878.-   16. Ang S L, Rossant J: HNF-3beta is essential for node and    notochord formation in mouse development. Cell 1994, 78:561-574.-   17. Kimura S et al.: The T/ebp null mouse: thyroid-specific    enhancer-binding protein is essential for the organogenesis of the    thyroid, lung, ventral forebrain, and pituitary. Genes Dev 1996,    10:60-69.-   18. Apelqvist A, Ahlgren U, Edlund H: Sonic hedgehog directs    specialized mesoderm differentiation in the intestine and pancreas.    Curr Biol 1997, 7:801-804.-   19. Chiang C et al.: Cyclopia and defective axial patterning in mice    lacking Sonic hedgehog gene function. Nature 1996, 383: 407-413.-   20. Chen H et al.: Limb and kidney defects in Lmx1b mutant mice    suggest and involvement of LMX1B in human nail patella syndrome.    Nature Genetics 1998, 19:51-55.

All of the above-cited references and publications are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention.

1. A method for modulating the growth state of lung tissue, or cellsderived therefrom, comprising ectopically contacting the tissue with anamount of an agent effective to alter the rate of proliferation of thelung tissue, wherein the agent is selected from the group consisting ofa hedgehog therapeutic, a ptc therapeutic and an fgf-10 therapeutic. 2.A method for inducing the formation of, or the maintenance or function aperformance of lung tissue, comprising contacting the lung tissue withan amount of an agent effective to induce the formation of new lungtissue, wherein the agent is selected from the group consisting of ahedgehog therapeutic, a ptc therapeutic and an fgf-10 therapeutic. 3.The method of claim 1, wherein the lung tissue is in culture, and theagent is provided as a cell culture additive.
 4. The method of claim 1,wherein the cell is treated in an animal and the agent is administeredto the animal as a therapeutic composition.
 5. The method of claim 1,wherein the agent is a hedgehog therapeutic.
 6. The method of claim 5,wherein the hedgehog therapeutic is a polypeptide including a hedgehogpolypeptide sequence of at least a bioactive extracellular portion of ahedgehog protein.
 7. The method of claim 6, wherein the polypeptideincludes at least 50 amino acids residues of an N-terminal half of thehedgehog protein
 8. The method of claim 6, wherein the polypeptideincludes at least 100 amino acids of an extracellular domain of thehedgehog protein.
 9. The method of claim 6, wherein the polypeptideincludes at least a portion of the hedgehog protein corresponding to a19 kd fragment of an extracellular domain of the hedgehog protein. 10.The method of claim 6, wherein the hedgehog protein is encoded by a geneof a vertebrate organism.
 11. The method of claim 6, wherein thepolypeptide includes a hedgehog polypeptide sequence represented in thegeneral formula of SEQ ID No.
 21. 12. The method of claim 6, wherein thepolypeptide includes a hedgehog polypeptide sequence represented in thegeneral formula of SEQ ID No.
 22. 13. The method of claim 6, wherein thehedgehog protein is encoded by a human hedgehog gene.
 14. The method ofclaim 6, wherein the hedgehog polypeptide sequence is at least 60percent identical to an amino acid sequence of a hedgehog proteinselected from the group consisting of SEQ ID No:9, SEQ ID No:10, SEQ IDNo:11, SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15 and SEQ IDNo:16.
 15. The method of claim 6, wherein the hedgehog polypeptidesequence is encodable by a nucleotide sequence which hybridizes understringent conditions to a sequence selected from the group consisting ofSEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ IDNo:6, SEQ ID No:7 and SEQ ID No:8.
 16. The method of claim 6, whereinthe hedgehog polypeptide sequence is an amino acid sequence of ahedgehog protein selected from the group consisting of SEQ ID No:9, SEQID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo:15 and SEQ ID No:16.
 17. The method of claim 6, wherein the hedgehogpolypeptide sequence is an amino acid sequence of a Sonic hedgehogprotein.
 18. The method of claim 1, wherein the agent is a ptctherapeutic.
 19. The method of claim 18, wherein the ptc therapeutic isa small organic molecule which binds to a patched protein andderepresses patched-mediated inhibition of mitosis.
 20. The method ofclaim 18, wherein the ptc therapeutic binds to patched and mimicshedgehog-mediated patched signal transduction.
 21. The method of claim20, wherein the ptc therapeutic is a small organic molecule.